Reactive Center Loop Research Articles

Effect of reactive center loop structure of antichymotrypsin on inhibition of duodenase activity

Interaction between duodenase (a granase family member) from bovine duodenal mucosa and recombinant antichymotrypsin (rACT) and its P1 variants has been studied. Association rate constants (k(a)) were 11, 6.8, and 17 mM(-1).sec(-1) for rACT, ACT L358M, and ACT L358R, respectively. Natural antitrypsin (AT) compared to ACT was a 20 times more effective duodenase inhibitor (in terms of k(a)). Duodenase interacted with P1 variants of ACT via a suicide mechanism with stoichiometry of the process SI = 1.2. The nature of the P1 residue of the inhibitor did not influence the interaction if other residues did not meet conformational requirements of the duodenase substrate-binding pocket. Also, interaction of duodenase with ACT variants containing residues from AT reaction center loop (rACT P2-P3', rACT P3-P4', rACT P4-P3', and rACT P6-P4') was studied. The inhibition type ([E](0) = 1.10(-7) M, 25 degrees C) was revealed to be reversible-like, and efficacy of inhibition decreased with increase in the substituted part of the reactive center loop. Constants of inhibition (K(i)) were measured. Efficacy of interaction between the enzyme (duodenase) and inhibitor depends on topochemical correspondence between a substrate-binding pocket of the enzyme and substrate structure.

I440V mutation in C1 esterase inhibitor gene in a patient with hereditary angioedema and its influence to the structure of C1 esterase inhibitor

Objective To assess the mutation in exon 8 of C1 esterase inhibitor （C1INH） gene in a patient with hereditary angioedema （HAE）. Methods Genomic DNA was extracted from a female patient with HAE as well as her mother and a normal human control. The fragment of exon 8 of C1INH gene was amplified by PCR and inserted into plasmid carrier pUC19 with the help of ligase. Then, the recombinant plasmid was transformed into competent cells of E. coli TG1 strains. After culture of positive transformant, plasmid DNA was extracted and subjected to sequencing. SDS-PAGE and Western blot were performed on the sera of the patient to detect the concentration and function of C1INH protein. Results An A1677G mutation at exon 8 of C1INH gene, which resulted in a substitution of isoleucine to valine at codon 440, was found in the patient who suffered from HAE type I. Additionally, SDS-PAGE and Western blot revealed that the molecular weight of CIINH protein was 96 000, but not 105 000 observed in normal human control. Conclusion The newly identified mutation I440V, which is located at P4 residue of reactive center loop in CIINH, may result in conformational alteration of C1INH. Key words: Angioneurotic edema; C1 esterase inhibitors; Variation （genetics）

Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α1-Antitrypsin Variants

BackgroundProteolytic processing of the Lassa virus envelope glycoprotein precursor GP-C by the host proprotein convertase site 1 protease (S1P) is a prerequisite for the incorporation of the subunits GP-1 and GP-2 into viral particles and, hence, essential for infectivity and virus spread. Therefore, we tested in this study the concept of using S1P as a target to block efficient virus replication.Methodology/Principal FindingWe demonstrate that stable cell lines inducibly expressing S1P-adapted α1-antitrypsin variants inhibit the proteolytic maturation of GP-C. Introduction of the S1P recognition motifs RRIL and RRLL into the reactive center loop of α1-antitrypsin resulted in abrogation of GP-C processing by endogenous S1P to a similar level observed in S1P-deficient cells. Moreover, S1P-specific α1-antitrypsins significantly inhibited replication and spread of a replication-competent recombinant vesicular stomatitis virus expressing the Lassa virus glycoprotein GP as well as authentic Lassa virus. Inhibition of viral replication correlated with the ability of the different α1-antitrypsin variants to inhibit the processing of the Lassa virus glycoprotein precursor.Conclusions/SignificanceOur data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies.

Local environment perturbations in alpha1-antitrypsin monitored by a ratiometric fluorescent label

The complex multistep inhibition of proteinases by alpha(1)-antitrypsin (alpha(1)-AT) was investigated by covalently labeling its unique Cys residue with a ratiometric environment-sensitive fluorescent dye, 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone (BMFC). The binding of BMFC-labeled alpha(1)-AT with pancreatic elastase led to significant changes in the dual emission of BMFC. The 8 nm blue shift of one of the bands and ca. 65% change in the intensity ratio of the two emission bands suggested an increased exposure of the labeled Cys-232 residue to the bulk water on complex formation. In contrast, the bacterial V8 proteinase-induced cleavage of the reactive center loop of BMFC-labeled alpha(1)-AT did not generate any significant change in the Cys-232 region. Similar experiments with elastase and alpha(1)-AT conjugated to the classical environment-sensitive dye, IANBD, confirmed these results but led to much smaller modifications in the emission spectrum. Stopped-flow investigation of the reaction between BMFC-labeled alpha(1)-AT and elastase showed both a well-described fast and a new slow step of the inhibition process. The latter step is probably associated with the structural reorganization aimed at stabilizing the final complex. These results present a convenient fluorescence ratiometric approach based on the BMFC label for studies of protein conformational changes.

1-antitrypsin and the maintenance of hemostatic balance

Serine protease inhibitors, known as serpins, belong to a superfamily of proteins that are the quintessential regulators of extracellular peptidase pathways.1 Serpins prevent the deleterious effects of excessive peptide bond hydrolysis. The principal determinant of serpin specificity appears to be the P1 residue localized within the substrate recognition sequence of the reactive center loop.2 A serine protease and its specific serpin form a tight inactive complex after cleavage of a scissile bond between P1 and P1’ residues. Antithrombin is the principal serpin involved in the regulation of blood clotting proteinases but it differs from other serpins in having several multiple target proteinases such as thrombin, factor Xa, factor IXa, factor XIa and factor XIIa. Its importance as a major regulator of thrombin generation is well demonstrated since the initial publication of antithrombin deficiency.3 Alpha1-antitrypsin has major homology with antithrombin, but its major substrate is the leucocyte elastase, with accessory inhibitory activity toward trypsin, chymotrypsin and activated protein C. P1-P1’ residues in antithrombin are constituted of the Arg 393-Ser 394 residues when this bound is Met 358-Ser 359 in α1-antitrypsin.

Exosites mediate the anti‐inflammatory effects of a multifunctional serpin from the saliva of the tick Ixodes ricinus

Serine protease inhibitors (serpins) are a structurally related but functionally diverse family of ubiquitous proteins. We previously described Ixodes ricinus immunosuppressor (Iris) as a serpin from the saliva of the tick I. ricinus displaying high affinity for human leukocyte elastase. Iris also displays pleotropic effects because it interferes with both the immune response and hemostasis of the host. It thus inhibits lymphocyte proliferation and the secretion of interferon-gamma or tumor necrosis factor-alpha by peripheral blood mononuclear cells, and also platelet adhesion, coagulation and fibrinolysis. Its ability to interfere with coagulation and fibrinolysis, but not platelet adhesion, depends on the integrity of its antiproteolytic reactive center loop domain. Here, we dissect the mechanisms underlying the interaction of recombinant Iris with peripheral blood mononuclear cells. We show that Iris binds to monocytes/macrophages and inhibits their ability to secrete tumor necrosis factor-alpha. Recombinant Iris also has a protective role in endotoxemic shock. The anti-inflammatory ability of Iris does not depend on its antiprotease activity. Moreover, we pinpoint the exosites involved in this activity.

The role of strand 1 of the C beta-sheet in the structure and function of alpha(1)-antitrypsin.

Serpins inhibit cognate serine proteases involved in a number of important processes including blood coagulation and inflammation. Consequently, loss of serpin function or stability results in a number of disease states. Many of the naturally occurring mutations leading to disease are located within strand 1 of the C beta-sheet of the serpin. To ascertain the structural and functional importance of each residue in this strand, which constitutes the so-called distal hinge of the reactive center loop of the serpin, an alanine scanning study was carried out on recombinant alpha(1)-antitrypsin Pittsburgh mutant (P1 = Arg). Mutation of the P10' position had no effect on its inhibitory properties towards thrombin. Mutations to residues P7' and P9' caused these serpins to have an increased tendency to act as substrates rather than inhibitors, while mutations at P6' and P8' positions caused the serpin to behave almost entirely as a substrate. Mutations at the P6' and P8' residues of the C beta-sheet, which are buried in the hydrophobic core in the native structure, caused the serpin to become highly unstable and polymerize much more readily. Thus, P6' and P8' mutants of alpha(1)-antitrypsin had melting temperatures 14 degrees lower than wild-type alpha(1)-antitrypsin. These results indicate the importance of maintaining the anchoring of the distal hinge to both the inhibitory mechanism and stability of serpins, the inhibitory mechanism being particularly sensitive to any perturbations in this region. The results of this study allow more informed analysis of the effects of mutations found at these positions in disease-associated serpin variants.

Characterization of Critical Residues of the Granzyme B Inhibitor, Serpina3n (50.43)

Abstract Granzyme B (GrB) is crucial for the immune system in eliminating tumor and virus-infected cells by caspase-mediated apoptosis. GrB also has detrimental effects in allograft tissue rejection and autoimmune diseases. Our aim was to design effective GrB inhibitors as unique immunosuppressive drugs in treating these disorders. Our lab has defined a novel murine inhibitor of GrB named serpina3n (ser3n). Ser3n is a superior candidate for drug design since it is the only known GrB inhibitor that is secreted extracellularly, making it more plasma stable. We performed scanning mutagenesis of the reactive centre loop (RCL) domain, which confers serpins with their protease specificity, to test which residues have inhibitory properties. Using an in vitro transcribed/translated (IVTT) ser3n protein, we found that the putative P1 residue (Met) and the P3' residue (Lys) are important for ser3n binding to GrB. Human antichymotrypsin (ACT) is highly homologous to ser3n, but is not a GrB inhibitor. Our goal was to generate a human secretory protein that inhibits GrB. We created an ACT-ser3n chimera by replacing the RCL of ACT with that of ser3n. We then created a mutant ACT-ser3n chimera with a Met-to-Asp mutation that resulted in greater GrB binding than the original chimera, to a degree equal to ser3n binding. This inhibitor will work on pathogenic states when secreted GrB is important. This reveals a potential drug for blocking GrB in autoimmune diseases.

Regulation of Manduca sexta hemolymph proteinase 8 by serpin‐1 isoforms

Some insect immune responses, including prophenoloxidase activation and Toll pathway initiation, are mediated by serine proteinase cascades and regulated by serpins. Hemolymph proteinase 8 (HP8), a trypsin‐like serine proteinase, is involved in a Toll‐like pathway by cleaving pro‐sp ?tzle, the putative ligand of M. sexta Toll. The M. sexta serpin‐1 gene is expressed as 12 isoforms, which vary in their reactive center loop due to alternative splicing of exon 9. Serpin‐1 isoforms A, E, and J can all inhibit a fungal trypsin‐like protease, PR2, in vitro. To identify the roles of serpins in the Toll pathway in M. sexta, we purified recombinant serpin‐1A, ‐1E, ‐1J and a mutant HP8 (proHP8Xa) in which the activation site was changed from NNDH to IEGR, allowing activation by bovine Factor Xa. Recombinant serpin‐1A, ‐1E, and ‐1J formed SDS‐stable complexes with HP8Xa. Serpin‐1J was the most effective inhibitor of HP8 among the tested serpin‐1 isoforms. The association rate constant of serpin‐1J and HP8 was 1.3×104 M‐1s‐1, indicating that serpin‐1J may contribute to the inhibitory regulation of HP8 in the hemolymph. Injection of serpin‐1J into M. sexta larvae resulted in a significant decrease in bacteria‐induced mRNA levels for the antibacterial peptides attacin and cecropin. These results suggest that serpin‐1J regulates the activity of HP8 to modulate the Toll pathway response in M. sexta. Supported by NIH grant GM41247.

Molecular Cloning and Biochemical Characterization of Manduca sexta Serpin‐7, a Regulator of Prophenoloxidase Activation

Serine protease inhibitors (serpins) are involved in various physiological reactions in humans and insects. Six serpins, SPN1‐SPN6, have been identified and characterized in the tobacco hornworm Manduca sexta. In this study, we obtained a complete DNA sequence of another Manduca serpin gene, SPN7. The putative mature SPN7 protein was 385 amino acid residues long (MW 42.90 kDa, pI 5.32) with a predicted 15 residue signal sequence. Multiple sequence alignment of the reactive center loop region of the M. sexta serpins indicated that SPN7 contained Arg and Ile residues at P1′‐P1 positions, similar to SPN4 and 5. RT‐PCR results demonstrated an up‐regulation of SPN7 expression in the fat body at 24 h after injection of larvae with Micrococcus. The recombinant SPN7 protein was expressed in E. coli and purified using nickel‐affinity chromatography. Addition of recombinant SPN7 significantly inhibited both 1) spontaneous melanization of the 5th instar larval plasma, and 2) phenoloxidase activity of the plasma challenged by Micrococcus bacteria. SPN7 inhibited the enzymatic activity of the active prophenoloxidase‐activating protease‐3 by forming a stable serpin‐protease complex as indicated by SDS‐PAGE under reducing conditions. SPN7 inhibited bovine trypsin but not chymotrypsin. We conclude that SPN7 is inhibitory and likely involved in regulation of innate immunity in the insect. (supported by NIH grant GM41247)

The 2.1-Å Crystal Structure of Native Neuroserpin Reveals Unique Structural Elements That Contribute to Conformational Instability

Neuroserpin is a selective inhibitor of tissue-type plasminogen activator (tPA) that plays an important role in neuronal plasticity, memory, and learning. We report here the crystal structure of native human neuroserpin at 2.1 A resolution. The structure has a helical reactive center loop and an omega loop between strands 1B and 2B. The omega loop contributes to the inhibition of tPA, as deletion of this motif reduced the association rate constant with tPA by threefold but had no effect on the kinetics of interaction with urokinase. Point mutations in neuroserpin cause the formation of ordered intracellular polymers that underlie dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type neuroserpin is also unstable and readily forms polymers under near-physiological conditions in vitro. This is, in part, due to the substitution of a conserved alanine for serine at position 340. The replacement of Ser340 by Ala increased the melting temperature by 3 degrees C and reduced polymerization as compared to wild-type neuroserpin. Similarly, neuroserpin has Asn-Leu-Val at the end of helix F and thus differs markedly from the Gly-X-Ile consensus sequence of the serpins. Restoration of these amino acids to the consensus sequence increased thermal stability and reduced the polymerization of neuroserpin and its transition to the latent conformer. Moreover, introduction of the consensus sequence into S49P neuroserpin that causes FENIB increased the stability and inhibitory activity of the mutant, as well as blocked polymerization and increased the yield of protein during refolding. These data provide a molecular explanation for the inherent instability of neuroserpin and the effect of point mutations that underlie the dementia FENIB.

Conformational Change in the Chromatin Remodelling Protein MENT

Chromatin condensation to heterochromatin is a mechanism essential for widespread suppression of gene transcription, and the means by which a chromatin-associated protein, MENT, induces a terminally differentiated state in cells. MENT, a protease inhibitor of the serpin superfamily, is able to undergo conformational change in order to effect enzyme inhibition. Here, we sought to investigate whether conformational change in MENT is ‘fine-tuned’ in the presence of a bound ligand in an analogous manner to other serpins, such as antithrombin where such movements are reflected by a change in intrinsic tryptophan fluorescence. Using this technique, MENT was found to undergo structural shifts in the presence of DNA packaged into nucleosomes, but not naked DNA. The contribution of the four Trp residues of MENT to the fluorescence change was mapped using deconvolution analysis of variants containing single Trp to Phe mutations. The analysis indicated that the overall emission spectra is dominated by a helix-H tryptophan, but this residue did not dominate the conformational change in the presence of chromatin, suggesting that other Trp residues contained in the A-sheet and RCL regions contribute to the conformational change. Mutagenesis revealed that the conformational change requires the presence of the DNA-binding ‘M-loop’ and D-helix of MENT, but is independent of the protease specificity determining ‘reactive centre loop’. The D-helix mutant of MENT, which is unable to condense chromatin, does not undergo a conformational change, despite being able to bind chromatin, indicating that the conformational change may contribute to chromatin condensation by the serpin.

Mutagenesis studies toward understanding the intracellular signaling mechanism of antithrombin.

Recent studies have indicated that antithrombin (AT) possesses both anti-inflammatory and antiangiogenic properties. The purpose of this study was to investigate the mechanism of the intracellular signaling activities of AT using wild-type and mutant serpins that have reduced anticoagulant activities due to mutations in either the reactive center loop (RCL) or the heparin-binding site. Direct cellular effects of the AT derivatives were compared in the LPS-stimulated endothelial cells by employing permeability and neutrophil adhesion assays in the absence and presence of pertussis toxin (PTX) and siRNAs for either syndecan-4 or sphingosine 1-phosphate receptor 1 (S1P(1)). Furthermore, the roles of prostacyclin and nuclear factor (NF)-kappaB in modulating these effects were investigated. Both wild-type and the RCL mutant, AT/Proth-2, exhibited similar potent barrier protective activities and inhibited the adhesion of neutrophils to endothelial cells via inhibition of the NF-kappaB pathway. Indomethacin abrogated both activities. The heparin-binding site mutants, AT-K114E and AT-K125E, did not exhibit any protective activity in either one of these assays, but a potent pro-apoptotic activity was observed for the AT-K114E in endothelial cells. Both PTX and siRNA for syndecan-4 inhibited the protective effect of AT, but the siRNA for S1P(1) was inconsequential. The interaction of AT with syndecan-4 is required for its prostacyclin-dependent protective effect through a PTX-sensitive and non-S1P(1)-related G(i)-protein coupled receptor. The RCL mutant, AT/Proth-2, with a markedly reduced anticoagulant but normal protective signaling properties, may potentially be developed as a safer anti-inflammatory drug without increasing the risk of bleeding.

Human Neuroserpin: Structure and Time-Dependent Inhibition

Human neuroserpin (hNS) is a protein serine protease inhibitor expressed mainly in the nervous system, where it plays key roles in neural development and plasticity by primarily targeting tissue plasminogen activator (tPA) . Four hNS mutations are associated to a form of autosomal dominant dementia, known as familial encephalopathy with neuroserpin inclusion bodies. The medical interest in and the lack of structural information on hNS prompted us to study the crystal structure of native and cleaved hNS, reported here at 3.15 and 1.85 Å resolution, respectively. In the light of the three-dimensional structures, we focus on the hNS reactive centre loop in its intact and cleaved conformations relative to the current serpin polymerization models and discuss the protein sites hosting neurodegenerative mutations. On the basis of homologous serpin structures, we suggest the location of a protein surface site that may stabilize the hNS native (metastable) form. In parallel, we present the results of kinetic studies on hNS inhibition of tPA. Our data analysis stresses the instability of the hNS–tPA complex with a dissociation half-life of minutes compared to a half-life of weeks observed for other serpin–cognate protease complexes.

Interactions of Plasminogen Activator Inhibitor-1 with Vitronectin Involve an Extensive Binding Surface and Induce Mutual Conformational Rearrangements

In order to explore early events during the association of plasminogen activator inhibitor-1 (PAI-1) with its cofactor vitronectin, we have applied a robust strategy that combines protein engineering, fluorescence spectroscopy, and rapid reaction kinetics. Fluorescence stopped-flow experiments designed to monitor the rapid association of PAI-1 with vitronectin indicate a fast, concentration-dependent, biphasic binding of PAI-1 to native vitronectin but only a monophasic association with the somatomedin B (SMB) domain, suggesting that multiple phases of the binding interaction occur only when full-length vitronectin is present. Nonetheless, in all cases, the initial fast interaction is followed by slower fluorescence changes attributed to a conformational change in PAI-1. Complementary experiments using an engineered, fluorescently silent PAI-1 with non-natural amino acids showed that concomitant structural changes occur as well in native vitronectin. Furthermore, we have measured the effect of vitronectin on the rate of insertion of the reactive center loop into beta-sheet A of PAI-1 during reaction with target proteases. With a variety of PAI-1 variants, we observe that both full-length vitronectin and the SMB domain have protease-specific effects on the rate of loop insertion but that the two exhibit clearly different effects. These results support a model for PAI-1 binding to vitronectin in which the interaction surface extends beyond the region of PAI-1 occupied by the SMB domain. In support of this model are recent results that define a PAI-1-binding site on vitronectin that lies outside the somatomedin B domain (Schar, C. R., Blouse, G. E., Minor, K. H., and Peterson, C. B. (2008) J. Biol. Chem. 283, 10297-10309) and the complementary site on PAI-1 (Schar, C. R., Jensen, J. K., Christensen, A., Blouse, G. E., Andreasen, P. A., and Peterson, C. B. (2008) J. Biol. Chem. 283, 28487-28496).

Conformational Dynamics of Antithrombin III With its Allosteric Activator Heparin

Antithrombin III (ATIII) is a serpin that is involved in the regulation of blood coagulation through the inhibition of blood clotting enzymes. Heparin is an allosteric activator of ATIII that binds to helix-D and causes a conformational change in the reactive center loop (RCL), expelling it from its position partially inserted into beta-sheet A. RCL expulsion in turn increases ATIII activity toward fXa several hundred-fold. Hydrogen/deuterium exchange and mass spectrometry were used to probe the dynamics of ATIII in the presence and absence of a synthetic heparin pentasaccharide (Fondaparinux). Results of our initial hydrogen/deuterium exchange mass spectrometry experiments provide direct, solution phase evidence that heparin cofactor binding alters conformational dynamics in four specific regions of the antithrombin molecule. (1) Helix D -- Heparin binding reduced H/D, consistent with hD extension upon cofactor binding. (2) Breach region -- Beta strands 3A and 5A, flanking the site for RCL insertion into sheet A, showed reduced H/D exchange, consistent with increased rigidity of the breach region and stabilization of a loop-expelled form. (3) Proximal RCL and hinge region -- H/D exchange for residues 376-387, which includes the N-terminal hinge region of the RCL, increased in the presence of heparin, indicating greater solvent exposure and expulsion from beta sheet A. (4) Distal RCL -- Deuterium exchange for 388-402, which includes s1C and the distal side of the RCL, decreased significantly in the presence of heparin, suggesting that s1C extension-mediated stabilization on the C-terminal side of RCL contributes to exposure of the proximal its end upon cofactor binding. Thus, dynamic H/D exchange studies of free and heparin-bound antithrombin molecules in solution will be useful for validating and refining models of ATIII heparin activation inferred from crystal structures.

A computational modeling and molecular dynamics study of the Michaelis complex of human protein Z-dependent protease inhibitor (ZPI) and factor Xa (FXa)

Protein Z-dependent protease inhibitor (ZPI) and antithrombin III (AT3) are members of the serpin superfamily of protease inhibitors that inhibit factor Xa (FXa) and other proteases in the coagulation pathway. While experimental structural information is available for the interaction of AT3 with FXa, at present there is no structural data regarding the interaction of ZPI with FXa, and the precise role of this interaction in the blood coagulation pathway is poorly understood. In an effort to gain a structural understanding of this system, we have built a solvent equilibrated three-dimensional structural model of the Michaelis complex of human ZPI/FXa using homology modeling, protein-protein docking and molecular dynamics simulation methods. Preliminary analysis of interactions at the complex interface from our simulations suggests that the interactions of the reactive center loop (RCL) and the exosite surface of ZPI with FXa are similar to those observed from X-ray crystal structure-based simulations of AT3/FXa. However, detailed comparison of our modeled structure of ZPI/FXa with that of AT3/FXa points to differences in interaction specificity at the reactive center and in the stability of the inhibitory complex, due to the presence of a tyrosine residue at the P1 position in ZPI, instead of the P1 arginine residue in AT3. The modeled structure also shows specific structural differences between AT3 and ZPI in the heparin-binding and flexible N-terminal tail regions. Our structural model of ZPI/FXa is also compatible with available experimental information regarding the importance for the inhibitory action of certain basic residues in FXa.

The Critical Role of Hinge-Region Expulsion in the Induced-Fit Heparin Binding Mechanism of Antithrombin

Antithrombin (AT) is the most important inhibitor of coagulation proteases. Its activity is stimulated by glycosaminoglycans, such as heparin, through allosteric and template mechanisms. AT utilises an induced-fit mechanism to bind with high affinity to a pentasaccharide sequence found in about one-third of heparin chains. The conformational changes behind this mechanism have been characterised by several crystal structures of AT in the absence and in the presence of pentasaccharide. Pentasaccharide binding ultimately results in a conformational change that improves affinity by about 1000-fold. Crystal structures show several differences, including the expulsion of the hinge region of the reactive centre loop from beta-sheet A, which is known to be critical for the allosteric activation of AT. Here, we present data that reveal an energetically distinct intermediate on the path to full activation where the majority of conformational changes have already occurred. A crystal structure of this intermediate shows that the hinge region is in a native-like state in spite of having the pentasaccharide bound in the normal fashion. We engineered a disulfide bond to lock the hinge in its native position to determine the energetic contributions of the initial and final conformational events. Approximately 60% of the free-energy contribution of conformational change is provided by the final step of hinge-region expulsion and subsequent closure of the main beta-sheet A. A new analysis of the individual structural changes provides a plausible mechanism for propagation of conformational change from the heparin binding site to the remote hinge region in beta-sheet A.

Crystal structure of SCCA1 and insight about the interaction with JNK1

Squamous cell carcinoma antigen 1 (SCCA1), which belongs to serine proteinase inhibitor (serpin) superfamily, inhibits papain-like cysteine proteinase. Recently, it has been reported that SCCA1 acts not only as a proteinase inhibitor but also as an inhibitor of UV-induced apoptosis via suppression of the activity of c-Jun NH 2-terminal kinase (JNK1). The present study determined the crystal structure of SCCA1, suggesting that the reactive center loop (RCL) of SCCA1, a recognition site of proteinase, is very flexible and located away form the main-body of SCCA1. We show that the inhibitory effect of SCCA1 on the kinase activity of JNK1 is lost when the RCL was truncated. Furthermore, we found that a mutant protein created by replacing one amino acid in RCL maintain the suppressive activity to JNK1, whereas the inhibitory effect to proteinase is obviously decreased.

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

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 beta-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 beta-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 beta-sheet A.