P1 variant antithrombins Glasgow (393 Arg to His) and Pescara (393 Arg to Pro) have increased heparin affinity and are resistant to catalytic cleavage by elastase Implications for the heparin activation mechanism

The heparin affinity of normal and two P1 variants of antithrombin-III (AT) was studied by gradient elution with NaCl in Tris buffer on heparin-Sepharose. At pH 7.4 normal AT eluted art [Na⁺] 0.78 mol/l and the variants both showed increased affinity with AT Pescara eluting at [Na⁺] 0.86 mol/l and AT Glasgow at [Na⁺] 0.92 mol/l. We have earlier proposed a model for heparin activation in which the native state of AT maintains a salt bridge involving the P1 Arg-393 residue. Binding of heparin induces a higher heparin affinity conformation in which the salt bridge is disrupted to reveal the reactive centre for inhibition of thrombin. The Glasgow and Pescara variants, lacking a reactive centre P1 basic residue, would be unable to form this salt bridge, and we suggested that the high affinity conformation which they adopt as their native state would resemble the heparin induced conformation. To examine this model, we measured the heparin induced fluorescence of two P1 variants and tested the susceptibility of their reactive loops to catalytic cleavage. Both variants had fluorescence spectra indistinguishable from normal AT. In the absence of heparin, neither variant was more susceptible than normal to catalytic cleavage by human neutrophil elastase. These findings suggest that the conformation of these P1 variants is different to that of fully heparinized normal AT.     

Canonical inhibitor-like interactions explain reactivity of alpha1-proteinase inhibitor Pittsburgh and antithrombin with proteinases

The serpin antithrombin is a slow thrombin inhibitor that requires heparin to enhance its reaction rate. In contrast, alpha1-proteinase inhibitor (alpha1PI) Pittsburgh (P1 Met --> Arg natural variant) inhibits thrombin 17 times faster than pentasaccharide heparin-activated antithrombin. We present here x-ray structures of free and S195A trypsin-bound alpha1PI Pittsburgh, which show that the reactive center loop (RCL) possesses a canonical conformation in the free serpin that does not change upon binding to S195A trypsin and that contacts the proteinase only between P2 and P2'. By inference from the structure of heparin cofactor II bound to S195A thrombin, this RCL conformation is also appropriate for binding to thrombin. Reaction rates of trypsin and thrombin with alpha1PI Pittsburgh and antithrombin and their P2 variants show that the low antithrombin-thrombin reaction rate results from the antithrombin RCL sequence at P2 and implies that, in solution, the antithrombin RCL must be in a similar canonical conformation to that found here for alpha1PI Pittsburgh, even in the nonheparin-activated state. This suggests a general, limited, canonical-like interaction between serpins and proteinases in their Michaelis complexes.     

The mechanism of high affinity pentasaccharide binding to antithrombin,insights from Gaussian accelerated molecular dynamics simulations

Abstract

The activity of antithrombin (AT), a serpin protease inhibitor, is enhanced by heparin and heparin analogs against its target proteases, mainly thrombin, factors Xa and IXa. Considerable amount of information is available on the multistep mechanism of the heparin pentasaccharide binding and conformational activation. However, much of the details were inferred from ‘static’ structures obtained by X-ray diffraction. Moreover, limited information is available for the early steps of binding mechanism other than kinetic studies with various ligands. To gain insights into these processes, we performed enhanced sampling molecular dynamics (MD) simulations using the Gaussian Accelerated Molecular Dynamics (GAMD) method, applied previously in drug binding studies. We were able to observe the binding of the pentasaccharide idraparinux to a ‘non-activated’ AT conformation in two separate trajectories with low root mean square deviation (RMSD) values compared to X-ray structures of the bound state. These trajectories along with further simulations of the AT-pentasaccharide complex provided insights into the mechanisms of multiple conformational transitions, including the expulsion of the hinge region, the extension of helix D and the conformational behavior of the reactive center loop (RCL). We could also confirm the high stability of helix P in non-activated AT conformations, such states might play an important role in heparin binding. ‘Generalized correlation’ matrices revealed possible paths of allosteric signal propagation to the binding sites for the target proteases, factors Xa and IXa. Enhanced MD simulations of ligand binding to AT may assist the design of new anticoagulant drugs.

Communicated by Ramaswamy H. Sarma     

Interaction between the P14 residue and strand 2 of beta-sheet B is critical for reactive center loop insertion in plasminogen activator inhibitor-2

The molecular interactions driving reactive center loop (RCL) insertion are of considerable interest in gaining a better understanding of the serpin inhibitory mechanism. Previous studies have suggested that interactions in the proximal hinge/breach region may be critical determinants of RCL insertion in serpins. In this study, conformational and functional changes in plasminogen activator inhibitor-2 (PAI-2) following incubation with a panel of synthetic RCL peptides indicated that the P14 residue is critical for RCL insertion, and hence inhibitory activity, in PAI-2. Only RCL peptides with a P14 threonine were able to induce the stressed to relaxed transition and abolish inhibitory activity in PAI-2, indicating that RCL insertion into beta-sheet A of PAI-2 is dependent upon this residue. The recently solved crystal structure of relaxed PAI-2 (PAI-2.RCL peptide complex) allowed detailed analysis of molecular interactions involving P14 related to RCL insertion. Of most interest is the rearrangement of hydrogen bonding around the breach region that accompanies the stressed to relaxed transition, in particular the formation of a side chain hydrogen bond between the threonine at P14 and an adjacent tyrosine on strand 2 of beta-sheet B in relaxed PAI-2. Structural alignment of known serpin sequences showed that this pairing (or the equivalent serine/threonine pairing) is highly conserved ( approximately 87%) in inhibitory serpins and may represent a general structural basis for serpin inhibitory activity.     

The conformational activation of antithrombin. A 2.85-A structure of a fluorescein derivative reveals an electrostatic link between the hinge and heparin binding regions

Antithrombin is unique among the serpins in that it circulates in a native conformation that is kinetically inactive toward its target proteinase, factor Xa. Activation occurs upon binding of a specific pentasaccharide sequence found in heparin that results in a rearrangement of the reactive center loop removing constraints on the active center P1 residue. We determined the crystal structure of an activated antithrombin variant, N135Q S380C-fluorescein (P14-fluorescein), in order to see how full activation is achieved in the absence of heparin and how the structural effects of the substitution in the hinge region are translated to the heparin binding region. The crystal structure resembles native antithrombin except in the hinge and heparin binding regions. The absence of global conformational change allows for identification of specific interactions, centered on Glu(381) (P13), that are responsible for maintenance of the solution equilibrium between the native and activated forms and establishes the existence of an electrostatic link between the hinge region and the heparin binding region. A revised model for the mechanism of the allosteric activation of antithrombin is proposed.     

Reactivities of the S2 and S3 subsite residues of thrombin with the native and heparin-induced conformers of antithrombin.

A pentasaccharide (PS) fragment of heparin capable of activating antithrombin (AT) markedly accelerates the inhibition of factor Xa by AT, but has insignificant effect on inhibition of thrombin. For inhibition of thrombin, the bridging function of a longer polysaccharide chain is required to accelerate the reaction. To study the basis for the similar reactivity of thrombin with the native or heparin-activated conformers of AT, several residues surrounding the active site pocket of thrombin were targeted for mutagenesis study. Leu99 and Glu192, the variant residues influencing the S2 and S3 subsite specificity of thrombin were replaced with Tyr and Gln. The Tyr60a, Pro60b, Pro60c, and Trp60d residues forming part of the S2 specificity pocket were deleted from the B-insertion loop of the wild-type and Leu99/Glu192 --> Tyr/Gln thrombins. Kinetic studies indicated that the reactivities of all mutants with AT were moderately or severely impaired. Although heparin largely corrected the defect in reactivities, it also markedly elevated the stoichiometries of inhibition with the mutants. Interestingly, PS also accelerated AT inhibition of the mutants 5-68-fold, suggesting that the mutants are able to discriminate between the native and activated conformers of AT. Based on these results and the recent crystal structure determination of AT in complex with PS, a model for thrombin-AT interaction is proposed in which the S2 and S3 subsite residues of thrombin are critical for recognition of the P2 and P3 residues of AT in the native conformation. In the activated conformation, other residues are made accessible for interaction with the protease, and the similar reactivity of thrombin with the native and heparin-activated conformers of AT may be coincidental. The results further suggest that the S2 and S3 subsite residues are crucial in controlling the partitioning of the thrombin-AT intermediate into the alternative inhibitory or substrate pathways of the reaction.     

The antithrombin P1 residue is important for target proteinase specificity but not for heparin activation of the serpin. Characterization of P1 antithrombin variants with altered proteinase specificity but normal heparin activation

Heparin has been proposed to conformationally activate the serpin, antithrombin, by making the reactive center loop P1 arginine residue accessible to proteinases. To evaluate this proposal, we determined the effect of mutating the P1 arginine on antithrombin's specificity for target and nontarget proteinases in both native and heparin-activated states of the serpin. As expected, mutation of the P1 arginine to tryptophan, histidine, leucine, and methionine converted the specificity of antithrombin from a trypsin inhibitor (k(assoc) = 2 x 10(5) M(-1) s(-1)) to a chymotrypsin inhibitor (k(assoc) = 10(3)-10(5) M(-1) s(-1)). However, heparin pentasaccharide activation increased the reactivity of the P1 variants with chymotrypsin or of the wild-type inhibitor with trypsin only 2-6-fold, implying that the P1 residue had similar accessibilities to these proteinases in native and activated states. Mutation of the P1 arginine greatly reduced k(assoc) for antithrombin inhibition of thrombin and factor Xa from 40- to 5000-fold, but heparin normally accelerated the reactions of the variant antithrombins with these enzymes to make them reasonably efficient inhibitors (k(assoc) = 10(3)-10(4) M(-1) s(-1)). Fluorescence difference spectra of wild-type and P1 tryptophan variant antithrombins showed that the P1 tryptophan exhibited fluorescence properties characteristic of a solvent-exposed residue which were insignificantly affected by heparin activation. Moreover, all P1 variant antithrombins bound heparin with approximately 2-3-fold higher affinities than the wild type. These findings are consistent with the P1 mutations disrupting a P1 arginine-serpin body interaction which stabilizes the native low-heparin affinity conformation, but suggest that this interaction is of low energy and unlikely to limit the accessibility of the P1 residue. Together, these findings suggest that the P1 arginine residue is similarly accessible to proteinases in both native and heparin-activated states of the serpin and contributes similarly to the specificity of antithrombin for thrombin and factor Xa in the two serpin conformational states. Consequently, determinants other than the P1 residue are responsible for enhancing the specificity of antithrombin for the two proteinases when activated by heparin.     

Full or partial substitution of the reactive center loop of alpha-1-proteinase inhibitor by that of heparin cofactor II: P1 Arg is required for maximal thrombin inhibition

The abundant plasma protein alpha(1)-proteinase inhibitor (alpha(1)-PI) physiologically inhibits neutrophil elastase (NE) and factor XIa and belongs to the serine protease inhibitor (serpin) protein superfamily. Inhibitory serpins possess a surface peptide domain called the reactive center loop (RCL), which contains the P1-P1' scissile peptide bond. Conversion of this bond in alpha(1)-PI from Met-Ser to Arg-Ser in alpha(1)-PI Pittsburgh (M358R) redirects alpha(1)-PI from inhibiting NE to inhibiting thrombin (IIa), activated protein C (APC), and other proteases. In contrast to either the wild-type or M358R alpha(1)-PI, heparin cofactor II (HCII) is a IIa-specific inhibitor with an atypical Leu-Ser reactive center. We examined the effects of replacement of all or part of the RCL of alpha(1)-PI with the corresponding parts of the HCII RCL on the activity and specificity of the resulting chimeric inhibitors. A series of 12 N-terminally His-tagged alpha(1)-PI proteins differing only in their RCL residues were expressed as soluble proteins in Escherichia coli. Substitution of the P16-P3' loop of alpha(1)-PI with that of HCII increased the low intrinsic antithrombin activity of alpha(1)-PI to near that of heparin-free HCII, while analogous substitution of the P2'-P3' dipeptide surpassed this level. However, gel-based complexing and quantitative kinetic assays showed that all mutant proteins inhibited thrombin at less than 2% of the rate of alpha(1)-PI (M358R) unless the P1 residue was also mutated to Arg. An alpha(1)-PI (P16-P3' HCII/M358R) variant was only 3-fold less active than M358R against IIa but 70-fold less active against APC. The reduction in anti-APC activity is desired in an antithrombotic agent, but the improvement in inhibitory profile came at the cost of a 3.5-fold increase in the stoichiometry of inhibition. Our results suggest that, while P1 Arg is essential for maximal antithrombin activity in engineered alpha(1)-PI proteins, substitution of the corresponding HCII residues can enhance thrombin specificity.     

Disruption of a tight cluster surrounding tyrosine 131 in the native conformation of antithrombin III activates it for factor Xa inhibition

The native conformation of antithrombin III (ATIII) is a poor inhibitor of its coagulation pathway target enzymes because of the partial insertion of its reactive center loop (RCL) in its central A beta-sheet. This study focused on tyrosine 131, which is located at the helix D-sheet A interface, adjacent to the ATIII pentasaccharide and heparin cofactor-binding sites and some 17A away from the RCL insertion. Crystallographic structures show that the Tyr(131) ring is buried in native ATIII and then becomes exposed when pentasaccharide binds to the inhibitor and activates it. This change suggested that Tyr(131) might serve as a switch for ATIII conformational activation. The hypothesis is supported by results from this study, which progressively removed atoms from the Tyr(131) side chain. Rates of heparin-independent Y131L and Y131A factor Xa inhibition were 25 and 29 times faster than for the control and Y131F, suggesting that Tyr(131) ring interactions with neighboring helix D and strand 2A residues shift the uncatalyzed native-to-activated conformational equilibrium toward the RCL-inserted state. Thermal denaturation experiments showed Y131A and Y131L were less stable than the control and Y131F, implying an increased tendency toward A-sheet mobility in these genetically activated molecules. Thus, the tight Tyr(131)-Asn(127)-Leu(130)-Leu(140)-Ser(142) cluster at the helix D-strand 2A interface of native antithrombin contributes significantly to the stability of the ground state conformation, and tyrosine 131 serves as a heparin-responsive molecular switch during the allosteric activation of ATIII anticoagulant activity.     

Antithrombin conformational modulation by D-myo-inositol 3,4,5,6-tetrakisphosphate (TMI), a novel scaffold for the development of antithrombotic agents

Antithrombin (AT) is a serpin that inhibits mainly thrombin and fXa after being activated by binding to glycosaminoglycans as heparin and heparan sulfate. Upon binding, the native AT conformation, relatively inactive as a protease inhibitor, is converted to an activated form. Recently, a new compound, named TMI, was discovered in our group with nanomolar affinity to antithrombin, and shown to be able to induce a partial activation of antithrombin. As TMI represents an original scaffold for structural optimizations aiming the development of new antithrombotic drugs, the present work demonstrated, through a series of molecular dynamics simulations, that TMI is able to modulate AT reactive center loop flexibility similarly to what is observed to heparin, as well as exposing AT P1 residue, Arg393. These results represent the first atomic level indication of AT conformational activation by TMI, and may offer a predictive basis for future studies aiming TMI structural optimization.     

Dynamic properties of the native free antithrombin from molecular dynamics simulations: computational evidence for solvent- exposed Arg393 side chain

While antithrombin (AT) has small basal inhibitory activity, it reaches its full inhibitory potential against activated blood coagulation factors, FXa, FIXa, and FIIa (thrombin), via an allosteric and/or template (bridging) mechanism by the action of heparin, heparan sulfate, or heparin-mimetic pentasaccharides (PS). From the numerous X-ray structures available for different conformational states of AT, only indirect and incomplete conclusions can be drawn on the inherently dynamic properties of AT. As a typical example, the basal inhibitory activity of AT cannot be interpreted on the basis of “non-activated” free antithrombin X-ray structures since the Arg393 side chain, playing crucial role in antithrombin-proteinase interaction, is not exposed. In order to reveal the intrinsic dynamic properties and the reason of basal inhibitory activity of antithrombin, 2 μs molecular dynamics simulations were carried out on its native free-forms. It was shown from the simulation trajectories that the reactive center loop which is functioning as “bait” for proteases, even without any biasing potential can populate conformational state in which the Arg393 side chain is solvent exposed. It is revealed from the trajectory analysis that the peptide sequences correspond to the helix D extension, and new helix P formation can be featured with especially large root-mean-square fluctuations. Mutual information analyses of the trajectory showed remarkable (generalized) correlation between those regions of antithrombin which changed their conformations as the consequence of AT–PS complex formation. This suggests that allosteric information propagation pathways are present even in the non-activated native form of AT.     

Depiction of the forces participating in the 2-O-sulfo-alpha-L-iduronic acid conformational preference in heparin sequences in aqueous solutions

2-O-Sulfo-alpha-l-iduronic acid (IdoA2S) is one of the main components of heparin, an anticoagulant and antithrombotic polysaccharide able to potentiate the inhibitory effect of antithrombin over plasma serine proteases. This monosaccharide unit adopts an equilibrium between chair (1C4) and skew-boat (2SO) forms as a function of heparin sequence size and composition. Although the prevalence of the 1C4 chair conformation in monosaccharides is understood, the reasons for the increase in 2SO contribution in the whole polysaccharide chain are still uncertain. In this context, 0.2 mus molecular dynamics simulations of IdoA2S-containing oligosaccharides indicated that stabilization due to intramolecular hydrogen bonds around IdoA2S is highly correlated (p0.001) with the expected conformational equilibrium for this residue in solution. This behavior explains the known effect of different heparin compositions, at the monosaccharide level, on IdoA2S conformation in biological solutions.     

Antithrombin: in control of coagulation

Antithrombin is a serine proteinase inhibitor (serpin) which controls the process of coagulation. It has a well defined structure, consisting of three beta-sheets, nine alpha-helices and a reactive centre loop (RCL). The RCL contains the reactive centre which harbours a bait sequence for target proteases; cleavage results in inhibition by a unique mechanism. The inhibitory activity of antithrombin is controlled by its interaction with the co-factor, heparin, which accelerates its interaction with target proteases. This ensures that heparin and its newer derivatives, such as heparin pentasaccharide, are the mainstay therapeutics for control of thrombosis or inappropriate clotting. The clinical importance of antithrombin is manifested by its clear association with thrombosis when deficiency states occur.     

The reactive-center loop of active PAI-1 is folded close to the protein core and can be partially inserted

Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of plasminogen activators and plays an important role in many pathophysiological processes. Like other members of the serpin family, PAI-1 has a reactive center consisting of a mobile loop (RCL) with P1 and P1' residues acting as a "bait" for cognate protease. In contrast to the other serpins, PAI-1 loses activity by spontaneous conversion to an inactive latent form. This involves full insertion of the RCL into beta-sheet A. To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by donor-donor energy migration and probe quenching methods reveal that the RCL is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants. Disulfide bonds can be formed in double-cysteine mutants with substitutions at positions P11 or P13 of the RCL and neighboring residues in beta-sheet A. This suggests that the RCL may be preinserted up to residue P13 in active PAI-1, and possibly even to residue P11. We propose that the close proximity of the RCL to the protein core, and the ability of the loop to preinsert into beta-sheet A is a possible reason for PAI-1 being able to convert spontaneously to its latent form.     

Transition of ovalbumin to thermostable structure entails conformational changes involving the reactive center loop

Ovalbumin is a serpin without inhibitory activity against proteases. During embryonic development, ovalbumin in the native (N) form undergoes changes and takes a heat-stable form, which was previously named HS-ovalbumin. It has been known that N-ovalbumin is artificially converted to another thermostable form called S-ovalbumin by heating at an alkaline pH. Here, we characterized further the three ovalbumin forms, N, HS, and S. The epitope of the monoclonal antibody 2B3/2H11, which recognizes N- and HS-ovalbumin but not S-ovalbumin, was found to reside in the region Glu-Val-Val-Gly-Ala-Ser-Glu-Ala-Gly-Val-Asp-Ala-Ala-Ser-Val-Ser-Glu-Glu-Phe-Arg, which corresponds to 340-359 of amino acid residues and is contained in the reactive center loop (RCL). Removal of RCL by elastase or subtilisin mitigated binding of the antibody. Dephosphorylation experiments indicated that the phosphorylated Ser-344 residue located on RCL is crucial for the epitope recognition. We suggest that the shift to the heat-stable form of ovalbumin accompanies a movement of RCL.     

Understanding the specificity of serpin–protease complexes through interface analysis

Serpins such as antithrombin, heparin cofactor II, plasminogen activator inhibitor, antitrypsin, antichymotrypsin, and neuroserpin are involved in important biological processes by inhibiting specific serine proteases. Initially, the protease recognizes the mobile reactive loop of the serpin eliciting conformational changes, where the cleaved loop together with the protease inserts into β-sheet A, translocating the protease to the opposite side of inhibitor leading to its inactivation. Serpin interaction with proteases is governed mainly by the reactive center loop residues (RCL). However, in some inhibitory serpins, exosite residues apart from RCL have been shown to confer protease specificity. Further, this forms the basis of multi-specificity of some serpins, but the residues and their dimension at interface in serpin-protease complexes remain elusive. Here, we present a comprehensive structural analysis of the serpin-protease interfaces using bio COmplexes COntact MAPS (COCOMAPS), PRotein Interface Conservation and Energetics (PRICE), and ProFace programs. We have carried out interface, burial, and evolutionary analysis of different serpin-protease complexes. Among the studied complexes, non-inhibitory serpins exhibit larger interface region with greater number of residue involvement as compared to the inhibitory serpins. On comparing the multi-specific serpins (antithrombin and antitrypsin), a difference in the interface area and residue number was observed, suggestive of a differential mechanism of action of these serpins in regulating their different target proteases. Further, detailed study of these multi-specific serpins listed few essential residues (common in all the complexes) and certain specificity (unique to each complex) determining residues at their interfaces. Structural mapping of interface residues suggested that individual patches with evolutionary conserved residues in specific serpins determine their specificity towards a particular protease.     

Structural and functional characterization of a highly specific serpin in the insect innate immunity

The Toll signaling pathway, an essential innate immune response in invertebrates, is mediated via the serine protease cascade. Once activated, the serine proteases are irreversibly inactivated by serine protease inhibitors (serpins). Recently, we identified three serpin-serine protease pairs that are directly involved in the regulation of Toll signaling cascade in a large beetle, Tenebrio molitor. Of these, the serpin SPN48 was cleaved by its target serine protease, Spätzle-processing enzyme, at a noncanonical P1 residue of the serpin''s reactive center loop. To address this unique cleavage, we report the crystal structure of SPN48, revealing that SPN48 exhibits a native conformation of human antithrombin, where the reactive center loop is partially inserted into the center of the largest β-sheet of SPN48. The crystal structure also shows that SPN48 has a putative heparin-binding site that is distinct from those of the mammalian serpins. Ensuing biochemical studies demonstrate that heparin accelerates the inhibition of Spätzle-processing enzyme by a proximity effect in targeting the SPN48. Our finding provides the molecular mechanism of how serpins tightly regulate innate immune responses in invertebrates.     

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. Figure Solvent equilibrated models for protein z-dependent protease inhibitor and its initial reactive complex with coagulation factor Xa (show here) are developed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. V.C. and C.J.L. contributed equally to this work. The solvent-equilibrated PDB structure of the ZPI/FXa will be made available upon request. Conflict of interest statement  The authors state that they have no conflict of interest.     

Structure of native protein C inhibitor provides insight into its multiple functions

Protein C inhibitor (PCI) is a multifunctional serpin with wide ranging protease inhibitory functions, unique cofactor binding activities, and potential non-inhibitory functions akin to the hormone-transporting serpins. To gain insight into the molecular mechanisms utilized by PCI we developed a robust expression system in Escherichia coli and solved the crystal structure of PCI in its native state. The five monomers obtained from our two crystal forms provide an NMR-like ensemble revealing regions of inherent flexibility. The reactive center loop (RCL) of PCI is long and highly flexible with no evidence of hinge region incorporation into beta-sheet A, as seen for other heparin-binding serpins. We adapted an extrinsic fluorescence method for determining dissociation constants for heparin and find that the N-terminal tail of PCI and residues adjacent to helix H are not involved in heparin binding. The minimal heparin length capable of tight binding to PCI was determined to be chains of eight monosaccharide units. A large hydrophobic pocket occupied by hydrophobic crystal contacts was found in an analogous position to the hormone-binding site in thyroxine-binding globulin. In conclusion, the data presented here provide important insights into the mechanisms by which PCI exercises its multiple inhibitory and non-inhibitory functions.     

Deletion of P1 arginine in a novel antithrombin variant (antithrombin London) abolishes inhibitory activity but enhances heparin affinity and is associated with early onset thrombosis

A novel variant of antithrombin, the major serpin inhibitor of coagulation proteases, has been identified in a patient with early onset thrombosis and abnormal plasma antithrombin activity. Sequencing of the antithrombin genes of the patient revealed that one of the two alleles was abnormal due to an in-frame deletion of the codon for the P1 arginine residue. The abnormal antithrombin was separated from the normal inhibitor by complexing the latter with thrombin followed by heparin-agarose affinity chromatography. The purified variant, antithrombin London, was completely inactive as a thrombin or factor Xa inhibitor even after heparin activation. Surprisingly, the variant bound heparin with a K(D) reflecting an approximately 10-fold greater affinity than the normal inhibitor. Stopped-flow kinetic analysis showed that this was almost entirely due to a more favorable conformational activation of the variant than the normal inhibitor, as reflected by a decreased rate constant for reversal of the activation. Consistent with its higher than normal heparin affinity, the inactive antithrombin variant was a potent competitive antagonist of the heparin-catalyzed reaction of normal antithrombin with thrombin but did not affect the uncatalyzed reaction. These results suggest that deletion of the antithrombin P1 residue partially activates the serpin by inducing strain in the reactive center loop, which destabilizes the native loop-buried state and favors the activated loop-exposed state with high heparin affinity. The unusually severe thrombosis associated with the heterozygous mutation may be explained by the ability of antithrombin London to bind endogenous heparan sulfate or heparin molecules with high affinity and to thereby block activation of the normal inhibitor.