Paramount Importance of Core Conformational Changes for Heparin Allosteric Activation of Antithrombin.

Antithrombin is unique among serpin family protein protease inhibitors with respect to the major reactive center loop (RCL) and core conformational changes that mediate allosteric activation of its anticoagulant function by heparin. A critical role for expulsion of the RCL hinge from a native stabilizing interaction with the hydrophobic core in the activation mechanism has been proposed from reports that antithrombin variants that block this change through engineered disulfide bonds block activation. However, the sufficiency of core conformational changes for activation without expulsion of the RCL from the core is suggested by variants that are activated without the need for heparin and retain the native RCL-core interaction.

Inhibitory serpins. New insights into their folding, polymerization, regulation and clearance.

Serpins are a widely distributed family of high molecular mass protein proteinase inhibitors that can inhibit both serine and cysteine proteinases by a remarkable mechanism-based kinetic trapping of an acyl or thioacyl enzyme intermediate that involves massive conformational transformation. The trapping is based on distortion of the proteinase in the complex, with energy derived from the unique metastability of the active serpin. Serpins are the favoured inhibitors for regulation of proteinases in complex proteolytic cascades, such as are involved in blood coagulation, fibrinolysis and complement activation, by virtue of the ability to modulate their specificity and reactivity.

The High Affinity Binding Site on Plasminogen Activator Inhibitor-1 (PAI-1) for the Low Density Lipoprotein Receptor-related Protein (LRP1) Is Composed of Four Basic Residues.

Plasminogen activator inhibitor 1 (PAI-1) is a serpin inhibitor of the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue plasminogen activator, which binds tightly to the clearance and signaling receptor low density lipoprotein receptor-related protein 1 (LRP1) in both proteinase-complexed and uncomplexed forms. Binding sites for PAI-1 within LRP1 have been localized to CR clusters II and IV. Within cluster II, there is a strong preference for the triple CR domain fragment CR456.

Saturation Mutagenesis of the Antithrombin Reactive Center Loop P14 Residue Supports a Three-step Mechanism of Heparin Allosteric Activation Involving Intermediate and Fully Activated States.

Past studies have suggested that a key feature of the mechanism of heparin allosteric activation of the anticoagulant serpin, antithrombin, is the release of the reactive center loop P14 residue from a native state stabilizing interaction with the hydrophobic core. However, more recent studies have indicated that this structural change plays a secondary role in the activation mechanism. To clarify this role, we expressed and characterized 15 antithrombin P14 variants.

Conformational activation of antithrombin by heparin involves an altered exosite interaction with protease.

Heparin allosterically activates antithrombin as an inhibitor of factors Xa and IXa by enhancing the initial Michaelis complex interaction of inhibitor with protease through exosites. Here, we investigate the mechanism of this enhancement by analyzing the effects of alanine mutations of six putative antithrombin exosite residues and three complementary protease exosite residues on antithrombin reactivity with these proteases in unactivated and heparin-activated states. Mutations of antithrombin Tyr(253) and His(319) exosite residues produced massive 10-200-fold losses in reactivity with factors Xa and IXa in both unactivated and heparin-activated states, indicating that these residues made critical attractive interactions with protease independent of heparin activation.

The allosteric mechanism of activation of antithrombin as an inhibitor of factor IXa and factor Xa: heparin-independent full activation through mutations adjacent to helix D.

Allosteric conformational changes in antithrombin induced by binding a specific heparin pentasaccharide result in very large increases in the rates of inhibition of factors IXa and Xa but not of thrombin. These are accompanied by CD, fluorescence, and NMR spectroscopic changes. X-ray structures show that heparin binding results in extension of helix D in the region 131-136 with coincident, and possibly coupled, expulsion of the hinge of the reactive center loop.

Kinetic intermediates en route to the final serpin-protease complex: studies of complexes of α1-protease inhibitor with trypsin.

Serpin protein protease inhibitors inactivate their target proteases through a unique mechanism in which a major serpin conformational change, resulting in a 70-Å translocation of the protease from its initial reactive center loop docking site to the opposite pole of the serpin, kinetically traps the acyl-intermediate complex. Although the initial Michaelis and final trapped acyl-intermediate complexes have been well characterized structurally, the intermediate stages involved in this remarkable transformation are not well understood. To better characterize such intermediate steps, we undertook rapid kinetic studies of the FRET and fluorescence perturbation changes of site-specific fluorophore-labeled derivatives of the serpin, α1-protease inhibitor (α1PI), which report the serpin and protease conformational changes involved in transforming the Michaelis complex to the trapped acyl-intermediate complex in reactions with trypsin.

Quantitative dissection of the binding contributions of ligand lysines of the receptor-associated protein (RAP) to the low density lipoprotein receptor-related protein (LRP1).

Although lysines are known to be critical for ligand binding to LDL receptor family receptors, relatively small reductions in affinity have been found when such lysines have been mutated. To resolve this paradox, we have examined the specific binding contributions of four lysines, Lys-253, Lys-256, Lys-270, and Lys-289, in the third domain (D3) of receptor-associated protein (RAP), by eliminating all other lysine residues. Using D3 variants containing lysine subsets, we examined binding to the high affinity fragment CR56 from LRP1.

How the serpin α1-proteinase inhibitor folds.

Serpins are remarkable and unique proteins in being able to spontaneously fold into a metastable conformation without the aid of a chaperone or prodomain. This metastable conformation is essential for inhibition of proteinases, so that massive serpin conformational change, driven by the favorable energetics of relaxation of the metastable conformation to the more stable one, can kinetically trap the proteinase-serpin acylenzyme intermediate. Failure to direct folding to the metastable conformation would lead to inactive, latent serpin.

A proximal pair of positive charges provides the dominant ligand-binding contribution to complement-like domains from the LRP (low-density lipoprotein receptor-related protein).

The LRP (low-density lipoprotein receptor-related protein) can bind a wide range of structurally diverse ligands to regions composed of clusters of ~40 residue Ca2+-dependent, disulfide-rich, CRs (complement-like repeats). Whereas lysine residues from the ligands have been implicated in binding, there has been no quantification of the energetic contributions of such interactions and hence of their relative importance in overall affinity, or of the ability of arginine or histidine residues to bind. We have used four representative CR domains from the principal ligand-binding cluster of LRP to determine the energetics of interaction with well-defined small ligands that include methyl esters of lysine, arginine, histidine and aspartate, as well as N-terminally blocked lysine methyl ester.

Crystal structure of plasminogen activator inhibitor-1 in an active conformation with normal thermodynamic stability.

The serpin plasminogen activator inhibitor-1 (PAI-1) is a crucial regulator in fibrinolysis and tissue remodeling. PAI-1 has been associated with several pathological conditions and is a validated prognostic marker in human cancers. However, structural information about the native inhibitory form of PAI-1 has been elusive because of its inherent conformational instability and rapid conversion to a latent, inactive structure.

Regulation of proteases by protein inhibitors of the serpin superfamily.

The serpins comprise an ancient superfamily of proteins, found abundantly in eukaryotes and even in some bacteria and archea, that have evolved to regulate proteases of both serine and cysteine mechanistic classes. Unlike the thermodynamically determined lock-and-key type inhibitors, such as those of the Kunitz and Kazal families, serpins use conformational change and consequent kinetic trapping of an enzyme intermediate to effect inhibition. By combining interactions of both an exposed reactive center loop and exosites outside this loop with the active site and complementary exosites on the target protease, serpins can achieve remarkable specificity.

Molecular mechanisms of antithrombin-heparin regulation of blood clotting proteinases. A paradigm for understanding proteinase regulation by serpin family protein proteinase inhibitors.

Serpin family protein proteinase inhibitors regulate the activity of serine and cysteine proteinases by a novel conformational trapping mechanism that may itself be regulated by cofactors to provide a finely-tuned time and location-dependent control of proteinase activity. The serpin, antithrombin, together with its cofactors, heparin and heparan sulfate, perform a critical anticoagulant function by preventing the activation of blood clotting proteinases except when needed at the site of a vascular injury. Here, we review the detailed molecular understanding of this regulatory mechanism that has emerged from numerous X-ray crystal structures of antithrombin and its complexes with heparin and target proteinases together with mutagenesis and functional studies of heparin-antithrombin-proteinase interactions in solution.

Basis for the specificity and activation of the serpin protein Z-dependent proteinase inhibitor (ZPI) as an inhibitor of membrane-associated factor Xa.

The serpin ZPI is a protein Z (PZ)-dependent specific inhibitor of membrane-associated factor Xa (fXa) despite having an unfavorable P1 Tyr. PZ accelerates the inhibition reaction approximately 2000-fold in the presence of phospholipid and Ca(2+). To elucidate the role of PZ, we determined the x-ray structure of Gla-domainless PZ (PZ(DeltaGD)) complexed with protein Z-dependent proteinase inhibitor (ZPI).

Activation of antithrombin as a factor IXa and Xa inhibitor involves mitigation of repression rather than positive enhancement.

Allosteric activation of antithrombin as a rapid inhibitor of factors IXa and Xa requires binding of a high-affinity heparin pentasaccharide. The currently-accepted mechanism involves removal of a constraint on the antithrombin reactive center loop (RCL) so that the proteinase can simultaneously engage both the P1 arginine and an exosite at Y253. Recent results suggest that this mechanism is incorrect in that activation can be achieved without loop expulsion, while the exosite can be engaged in both low and high activity states.

Specificity of binding of the low density lipoprotein receptor-related protein to different conformational states of the clade E serpins plasminogen activator inhibitor-1 and proteinase nexin-1.

The low density lipoprotein receptor-related protein (LRP) is the principal clearance receptor for serpins and serpin-proteinase complexes. The ligand binding regions of LRP consist of clusters of cysteine-rich approximately 40-residue complement-like repeats (CR), with cluster II being the principal ligand-binding region. To better understand the specificity of binding at different sites within the cluster and the ability of LRP to discriminate in vivo between uncomplexed and proteinase-complexed serpins, we have systematically examined the affinities of plasminogen activator inhibitor-1 (PAI-1) and proteinase nexin-1 (PN-1) in their native, cleaved, and proteinase-complexed states to (CR)(2) and (CR)(3) fragments of LRP cluster II.

Receptor-associated protein (RAP) has two high-affinity binding sites for the low-density lipoprotein receptor-related protein (LRP): consequences for the chaperone functions of RAP.

RAP (receptor-associated protein) is a three domain 38 kDa ER (endoplasmic reticulum)-resident protein that is a chaperone for the LRP (low-density lipoprotein receptor-related protein). Whereas RAP is known to compete for binding of all known LRP ligands, neither the location, the number of binding sites on LRP, nor the domains of RAP involved in binding is known with certainty. We have systematically examined the binding of each of the three RAP domains (D1, D2 and D3) to tandem and triple CRs (complement-like repeats) that span the principal ligand-binding region, cluster II, of LRP.

High-resolution structure of the stable plasminogen activator inhibitor type-1 variant 14-1B in its proteinase-cleaved form: a new tool for detailed interaction studies and modeling.

Wild-type plasminogen activator inhibitor type-1 (PAI-1) rapidly converts to the inactive latent state under conditions of physiological pH and temperature. For in vivo studies of active PAI-1 in cell culture and in vivo model systems, the 14-1B PAI-1 mutant (N150H-K154T-Q319L-M354I), with its stabilized active conformation, has thus become the PAI-1 of choice. As a consequence of the increased stability, the only two forms likely to be encountered are the active or the cleaved form, the latter either free or complexed with target proteinase.

alpha-Macroglobulins are present in some gram-negative bacteria: characterization of the alpha2-macroglobulin from Escherichia coli.

alpha-Macroglobulins (alphaMs) are large glycoproteins that have been identified in a wide range of vertebrate and invertebrate species and are mostly thiol ester containing proteinase inhibitors. A recent analysis of bacterial genomes ( Budd, A., Blandin, S.

Characterization of the conformational alterations, reduced anticoagulant activity, and enhanced antiangiogenic activity of prelatent antithrombin.

A conformationally altered prelatent form of antithrombin that possesses both anticoagulant and antiangiogenic activities is produced during the conversion of native to latent antithrombin (Larsson, H., Akerud, P., Nordling, K.

Human alpha2-macroglobulin is composed of multiple domains, as predicted by homology with complement component C3.

Human alpha2M (alpha2-macroglobulin) and the complement components C3 and C4 are thiol ester-containing proteins that evolved from the same ancestral gene. The recent structure determination of human C3 has allowed a detailed prediction of the location of domains within human alpha2M to be made. We describe here the expression and characterization of three alpha(2)M domains predicted to be involved in the stabilization of the thiol ester in native alpha2M and in its activation upon bait region proteolysis.

Maspin binds to urokinase-type and tissue-type plasminogen activator through exosite-exosite interactions.

Maspin is a member of the serpin family with a reactive center loop that is incompatible with proteinase inhibition by the serpin conformational change mechanism. Despite this there are reports that maspin might regulate uPA-dependent processes in vivo. Using exogenous and endogenous fluorescence, we demonstrate here that maspin can bind uPA and tPA in both single-chain and double-chain forms, with K(d) values between 300 and 600 nM.

Serine and cysteine proteases are translocated to similar extents upon formation of covalent complexes with serpins. Fluorescence perturbation and fluorescence resonance energy transfer mapping of the protease binding site in CrmA complexes with granzyme B and caspase-1.

CrmA is a "cross-class" serpin family inhibitor of the proapoptotic serine protease, granzyme B, as well as cysteine proteases of the caspase family. To determine whether crmA inhibits these structurally diverse proteases by a common conformational trapping mechanism, we mapped the position of the protease in crmA complexes with granzyme B or caspase-1 by fluorescence perturbation and fluorescence resonance energy transfer (FRET) analyses of site-specific fluorophore-labeled crmAs. A reactive loop P6 NBD label underwent similar large fluorescence enhancements (>200%) either upon reactive loop cleavage by AspN protease or complex formation with granzyme B or caspase-1, consistent with the insertion of the cleaved reactive loop into sheet A in both types of crmA-protease complexes.

Cytokine response modifier a inhibition of initiator caspases results in covalent complex formation and dissociation of the caspase tetramer.

Active caspases are generally composed of two catalytic domains, each containing a large (p20) and a small (p10) subunit so that a fully active caspase has the organization (p20-p10)(2). The cowpox serpin crmA suppresses host apoptosis and inflammation by inhibiting endogenous caspases. We report on the mechanism crmA uses to inhibit caspases 1, 6, and 8.