Helix D elongation and allosteric activation of antithrombin.

Antithrombin requires allosteric activation by heparin for efficient inhibition of its target protease, factor Xa. A pentasaccharide sequence found in heparin activates antithrombin by inducing conformational changes that affect the reactive center of the inhibitor resulting in optimal recognition by factor Xa. The mechanism of transmission of the activating conformational change from the heparin-binding region to the reactive center loop remains unresolved. To investigate the role of helix D elongation in the allosteric activation of antithrombin, we substituted a proline residue for Lys(133). Heparin binding affinity was reduced by 25-fold for the proline variant compared with the control, and a significant decrease in the associated intrinsic fluorescence enhancement was also observed. Rapid kinetic studies revealed that the main reason for the reduced affinity for heparin was an increase in the rate of the reverse conformational change step. The pentasaccharide-accelerated rate of factor Xa inhibition for the proline variant was 10-fold lower than control, demonstrating that the proline variant cannot be fully activated toward factor Xa. We conclude that helix D elongation is critical for the full conversion of antithrombin to its high affinity, activated state, and we propose a mechanism to explain how helix D elongation is coupled to allosteric activation.     

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. We propose a quite different mechanism in which heparin activates antithrombin by mitigating an unfavorable surface interaction, by altering its nature, and by moving the attached proteinase away from the site of the unfavorable interaction through RCL expulsion.     

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     

Kinetic evidence that allosteric activation of antithrombin by heparin is mediated by two sequential conformational changes

The serpin, antithrombin, requires allosteric activation by a sequence-specific pentasaccharide unit of heparin or heparan sulfate glycosaminoglycans to function as an anticoagulant regulator of blood clotting proteases. Surprisingly, X-ray structures have shown that the pentasaccharide produces similar induced-fit changes in the heparin binding site of native and latent antithrombin despite large differences in the heparin affinity and global conformation of these two forms. Here we present kinetic evidence for similar induced-fit mechanisms of pentasaccharide binding to native and latent antithrombins and kinetic simulations which together support a three-step mechanism of allosteric activation of native antithrombin involving two successive conformational changes. Equilibrium binding studies of pentasaccharide interactions with native and latent antithrombins and the salt dependence of these interactions suggest that each conformational change is associated with distinct spectroscopic changes and is driven by a progressively better fit of the pentasaccharide in the binding site. The observation that variant antithrombins that cannot undergo the second conformational change bind the pentasaccharide like latent antithrombin and are partially activated suggests that both conformational changes contribute to allosteric activation, in agreement with a recently proposed model of allosteric 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. To examine the importance of helix D extension, we have introduced strong helix-promoting mutations in the 131–136 region of antithrombin (YRKAQK to LEEAAE). The resulting variant has endogenous fluorescence indistinguishable from WT antithrombin yet, in the absence of heparin, shows massive enhancements in rates of inhibition of factors IXa and Xa (114- and 110-fold, respectively), but not of thrombin, together with changes in near- and far-UV CD and ¹H NMR spectra. Heparin binding gives only ∼3–4-fold further rate enhancement but increases tryptophan fluorescence by ∼23% without major additional CD or NMR changes. Variants with subsets of these mutations show intermediate activation in the absence of heparin, again with basal fluorescence similar to WT and large increases upon heparin binding. These findings suggest that in WT antithrombin there are two major complementary sources of conformational activation of antithrombin, probably involving altered contacts of side chains of Tyr-131 and Ala-134 with core hydrophobic residues, whereas the reactive center loop hinge expulsion plays only a minor additional role.     

Critical role of the linker region between helix D and strand 2A in heparin activation of antithrombin

The binding of pentasaccharide heparin to antithrombin induces a conformational change that is transmitted to the reactive center loop and increases the rate of inhibition of factor Xa by approximately 300-fold. The mechanism of such transmission is not known. To test the role of residues 134-137, which link helix D to beta-sheet A, in this signal transduction, we created variant antithrombins in which we removed amino acids 134-137 stepwise and cumulatively. Although the deletions did not compromise the fundamental ability of antithrombin to bind to heparin or to inhibit target proteinases thrombin and factor Xa, they did largely decouple conformational changes in the heparin-binding site from conformational activation of the reactive center loop. Because the variant with only Ala(134) removed was as compromised as variants with larger deletions, yet the variant with Ser(137) removed was normal, we concluded that the length of the linker is less important than the precise interrelationship between residues in this region and other residues involved in conformational activation of antithrombin.     

Allosteric activation of antithrombin critically depends upon hinge region extension

Antithrombin (AT) inhibits most of the serine proteases generated in the blood coagulation cascade, but its principal targets are factors IXa, Xa, and thrombin. Heparin binding to AT, via a specific pentasaccharide sequence, alters the conformation of AT in a way that promotes efficient inhibition of factors IXa and Xa, but not of thrombin. The conformational change most likely to be relevant to protease recognition is the expulsion of the N-terminal portion of the reactive center loop (hinge region) from the main beta-sheet A. Here we investigate the hypothesis that the exosites on the surface of AT are accessible for interaction with a protease only when the hinge region is fully extended, as seen in the related Michaelis complex between heparin cofactor II and thrombin. We engineered a disulfide bond between residues 222 on strand 3A and 381 in the reactive center loop to prevent the extension of the hinge region upon pentasaccharide binding. The disulfide bond did not significantly alter the ability of the variant to bind to heparin or to inhibit thrombin. Although the basal rate of factor Xa inhibition was not affected, that of factor IXa inhibition was reduced to the limit of detection. In addition, the disulfide bond completely abrogated the pentasaccharide accelerated inhibition of factors Xa and IXa. We conclude that AT hinge region extension is the activating conformational change for inhibition of factors IXa and Xa, and propose models for the progressive and activated AT Michaelis complexes with thrombin, factor Xa, and factor IXa.     

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.     

Importance of the P2 glycine of antithrombin in target proteinase specificity, heparin activation, and the efficiency of proteinase trapping as revealed by a P2 Gly --> Pro mutation.

A sequence-specific heparin pentasaccharide activates the serpin, antithrombin, to inhibit factor Xa through an allosteric mechanism, whereas full-length heparin chains containing this sequence further activate the serpin to inhibit thrombin by an alternative bridging mechanism. To test whether the factor Xa specificity of allosterically activated antithrombin is encoded in the serpin reactive center loop, we mutated the factor Xa-preferred P2 Gly to the thrombin-preferred P2 Pro. Kinetic studies revealed that the mutation maximally enhanced the reactivity of antithrombin with thrombin 15-fold and decreased its reactivity toward factor Xa 2-fold when the serpin was activated by heparin pentasaccharide, thereby transforming antithrombin into an allosterically activated inhibitor of both factor Xa and thrombin. Surprisingly, the enhanced thrombin specificity of the mutant antithrombin was attenuated when a full-length bridging heparin was the activator, due both to a reduced rate of covalent reaction of the mutant serpin and thrombin and preferred reaction of the mutant serpin as a substrate. These results demonstrate that the reactive center loop sequence determines the specificity of allosterically activated antithrombin for factor Xa and that the conformational flexibility of the P2 Gly may be critical for optimal bridging of antithrombin and thrombin by physiologic heparin and for preventing antithrombin from reacting as a substrate in the bridging complex.     

Heparin binding site, conformational change, and activation of antithrombin.

Alignment of the heparin-activated serpins indicates the presence of two binding sites for heparin: a small high-affinity site on the D-helix corresponding in size to the minimal pentasaccharide heparin, and a longer contiguous low-affinity site extending to the reactive center pole of the molecule. Studies of the complexing of antithrombin and its variants with heparin fractions and with reactive center loop peptides including intermolecular loop-sheet polymers all support a 3-fold mechanism for the heparin activation of antithrombin. Binding to the pentasaccharide site induces a conformational change as measured by circular dichroism. Accompanying this, the reactive center becomes more accessible to proteolytic cleavage and there is a 100-fold increase in the kass for factor Xa but only a 10-fold increase for thrombin, to 6.4 x 10(4) M-1 s-1. To obtain a 100-fold increase in the kass for thrombin requires in addition a 4:1 molar ratio of disaccharide to neutralize the charge on the extended low-affinity site. Full activation requires longer heparin chains in order to stabilize the ternary complex between antithrombin and thrombin. Thus, addition of low-affinity but high molecular weight heparin in conjunction with pentasaccharide gives an overall kass of 2.7 x 10(6) M-1 s-1, close to that of maximal heparin activation.     

Localization of an antithrombin exosite that promotes rapid inhibition of factors Xa and IXa dependent on heparin activation of the serpin

We have previously shown that exosites in antithrombin outside the P6-P3' reactive loop region become available upon heparin activation to promote rapid inhibition of the target proteases, factor Xa and factor IXa. To identify these exosites, we prepared six antithrombin-alpha 1-proteinase inhibitor chimeras in which antithrombin residues 224-286 and 310-322 that circumscribe a region surrounding the reactive loop on the inhibitor surface were replaced in 10-16-residue segments with the homologous segments of alpha1-proteinase inhibitor. All chimeras bound heparin with a high affinity similar to wild-type, underwent heparin-induced fluorescence changes indicative of normal conformational activation, and were able to form SDS-stable complexes with thrombin, factor Xa, and factor IXa and inhibit these proteases with stoichiometries minimally altered from those of wild-type antithrombin. With only one exception, conformational activation of the chimeras with a heparin pentasaccharide resulted in normal approximately 100-300-fold enhancements in reactivity with factor Xa and factor IXa. The exception was the chimera in which residues 246-258 were replaced, corresponding to strand 3 of beta-sheet C, which showed little or no enhancement of its reactivity with these proteases following pentasaccharide activation. By contrast, all chimeras including the strand 3C chimera showed essentially wild-type reactivities with thrombin after pentasaccharide activation as well as normal full-length heparin enhancements in reactivity with all proteases due to heparin bridging. These findings suggest that antithrombin exosites responsible for enhancing the rates of factor Xa and factor IXa inhibition in the conformationally activated inhibitor lie in strand 3 of beta-sheet C of the serpin.     

Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin

Heparin activates the primary serpin inhibitor of blood clotting proteinases, antithrombin, both by an allosteric conformational change mechanism that specifically enhances factor Xa inactivation and by a ternary complex bridging mechanism that promotes the inactivation of thrombin and other target proteinases. To determine whether the factor Xa specificity of allosterically activated antithrombin is encoded in the reactive center loop sequence, we attempted to switch this specificity by mutating the P6-P3' proteinase binding sequence excluding P1-P1' to a more optimal thrombin recognition sequence. Evaluation of 12 such antithrombin variants showed that the thrombin specificity of the serpin allosterically activated by a heparin pentasaccharide could be enhanced as much as 55-fold by changing P3, P2, and P2' residues to a consensus thrombin recognition sequence. However, at most 9-fold of the enhanced thrombin specificity was due to allosteric activation, the remainder being realized without activation. Moreover, thrombin specificity enhancements were attenuated to at most 5-fold with a bridging heparin activator. Surprisingly, none of the reactive center loop mutations greatly affected the factor Xa specificity of the unactivated serpin or the several hundred-fold enhancement in factor Xa specificity due to activation by pentasaccharide or bridging heparins. Together, these results suggest that the specificity of both native and heparin-activated antithrombin for thrombin and factor Xa is only weakly dependent on the P6-P3' residues flanking the primary P1-P1' recognition site in the serpin-reactive center loop and that heparin enhances serpin specificity for both enzymes through secondary interaction sites outside the P6-P3' region, which involve a bridging site on heparin in the case of thrombin and a previously unrecognized exosite on antithrombin in the case of factor Xa.     

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.     

Serine 380 (P14) --> glutamate mutation activates antithrombin as an inhibitor of factor Xa

Heparin regulates the inhibitory activity of antithrombin. It has been proposed that residues P15 and P14 are expelled from beta-sheet A of antithrombin by heparin binding, permitting better interaction of the reactive center loop with factor Xa. We have made a P14 antithrombin variant (S380E) to create an activated inhibitory form of antithrombin in which P14 is already expelled from beta-sheet A. S380E antithrombin fluorescence is enhanced 35 +/- 5% compared with control antithrombin. There is minimal further increase in antithrombin fluorescence upon heparin binding. The variant has a 5 degrees C lower T(m) than control antithrombin. The variant is an inhibitor of proteinases and has a nearly 200-fold increased basal rate of inhibition of factor Xa, after correction for an increased stoichiometry of inhibition. This is comparable to that of antithrombin activated by high affinity heparin pentasaccharide. Full-length high affinity heparin causes only a 7-fold additional increase in rate and a large increase in stoichiometry of inhibition. In contrast, the basal rate of inhibition of thrombin is similar to that of control antithrombin but is increased 300-fold by heparin. These findings suggest that the native state of the S380E variant exists in a loop-expelled conformation that is consequently highly reactive toward factor Xa.     

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.     

Conformational equilibrium of the reactive center loop of antithrombin examined by steady state and time-resolved fluorescence measurements: consequences for the mechanism of factor Xa inhibition by antithrombin-heparin complexes

Activation of antithrombin by high-affinity heparin as an inhibitor of factor Xa has been ascribed to an allosteric switch between two conformations of the reactive center loop. However, we have previously shown that other, weaker binding, charged polysaccharides can give intermediate degrees of activation [Gettins, P. G. W., et al. (1993) Biochemistry 32, 8385-8389]. To examine whether such intermediate activation results from different reactive center loop conformations or, more simply, from a different equilibrium constant between the same two extreme conformations, we have used NBD covalently bound at the P1 position of an engineered R393C variant of antithrombin as a fluorescent reporter group and measured fluorescence lifetimes of the label in free antithrombin as well as in antithrombin saturated with long-chain high-affinity heparin, high-affinity heparin pentasaccharide, long-chain low-affinity heparin, and dextran sulfate. Steady state emission spectra, anisotropies, and dynamic quenching measurements were also recorded. We found that the large steady state fluorescence enhancements produced by binding of activators resulted from relief of a static quench of fluorescence of NBD in approximately 50% of the labeled antithrombin molecules rather than from any large change in lifetimes, and that similar lifetimes were found for NBD in all activated antithrombin-oligosaccharide complexes. Similar anisotropies and positions of the NBD emission maxima were also found in the absence and presence of activators. In addition, NBD was accessible to quenching agents in both the absence and presence of activators, with an at most 2-fold increase in quenching constants between these two extremes. The simplest interpretation of the partial static quench in the absence of activators, the different degrees of enhancement by different antithrombin activators, and the similar fluorescence properties and quenching behavior of the different states is that there are two distinct types of conformational equilibrium involving three distinct states of antithrombin, which we designate A, A', and B. A and A' represent low-affinity or inactive states of approximately equal energy, both having the hinge residues inserted into beta-sheet A. A is fluorescent, while A' is statically quenched. State B represents the activated loop-expelled conformation in which none of the NBD fluorophores are statically quenched, as a result of the loop, including the P1-NBD, moving away from the body of the antithrombin. Different activators are able to shift the equilibrium to the high-activity (B) state to different extents and hence give different degrees of measured activity, and different degrees of relief of static quench. The similar properties and accessibility of the NBD in the A and B conformations also indicate that the P1 side chain is not buried in the low-activity A conformation, suggesting that an earlier proposal that activation involves exposure of the P1 side chain cannot be the explanation for activation. As an alternative explanation, heparin activation may give access to an exosite on antithrombin for binding to factor Xa and hence be the principal basis for enhancement of the rate of inhibition.     

The influence of hinge region residue Glu-381 on antithrombin allostery and metastability

Antithrombin becomes an efficient inhibitor of factor Xa and thrombin by binding a specific pentasaccharide sequence found on a small fraction of the heparan sulfate proteoglycans lining the microvaculature. In the structure of native antithrombin, the reactive center loop is restrained due to the insertion of its hinge region into the main beta-sheet A, whereas in the heparin-activated state the reactive center loop is freed from beta-sheet A. In both structures, hinge region residue Glu-381 makes several stabilizing contacts. To determine the role of these contacts in the allosteric mechanism of antithrombin activation, we replaced Glu-381 with an alanine. This variant is less active toward its target proteases than control antithrombin, due to a perturbation of the equilibrium between the two forms, and to an increase in stoichiometry of inhibition. Pentasaccharide binding affinity is reduced 4-fold due to an increase in the off-rate. These data suggest that the main role of Glu-381 is to stabilize the activated conformation. Stability studies also showed that the E381A variant is resistant to continued insertion of its reactive center loop upon incubation at 50 degrees C, suggesting new stabilizing interactions in the native structure. To test this hypothesis, and to aid in the interpretation of the kinetic data we solved to 2.6 A the structure of the variant. We conclude that wild-type Glu-381 interactions stabilize the activated state and decreases the energy barrier to full loop insertion.     

Examination, by 1H-n.m.r. spectroscopy, of the binding of a synthetic, high-affinity heparin pentasaccharide to human antithrombin III

Binding of a synthetic, high-affinity heparin pentasaccharide and of intact heparin to both native and elastase-modified human antithrombin III have been examined by 1H-n.m.r. spectroscopy. The pentasaccharide perturbs many protein resonances in the same way as does intact heparin. There are, however, differences that seem to arise both from fewer contacts in the heparin binding-site when the pentasaccharide binds and from dissimilar conformational changes in the protein. The resonance of the H-2 atom of the histidine, considered to be the N-terminal residue and to be located in the heparin binding-site, is strongly perturbed by heparin binding both to native and modified antithrombin. The pentasaccharide has little effect on this histidine in either protein. Resonances from two of the remaining four histidine units are sensitive to longer-range conformational changes, and show differences between binding of the two heparin species both in native and modified ATIII. It is concluded that the pentasaccharide only partly fills the heparin binding-site and does not produce a conformational change identical to that caused by intact heparin. This is particularly significant as regards the mechanism of action of heparin, because the synthetic pentasaccharide activates ATIII towards Factor Xa, but not towards thrombin.     

The role of Arg46 and Arg47 of antithrombin in heparin binding.

Heparin greatly accelerates the reaction between antithrombin and its target proteinases, thrombin and factor Xa, by virtue of a specific pentasaccharide sequence of heparin binding to antithrombin. The binding occurs in two steps, an initial weak interaction inducing a conformational change of antithrombin that increases the affinity for heparin and activates the inhibitor. Arg46 and Arg47 of antithrombin have been implicated in heparin binding by studies of natural and recombinant variants and by the crystal structure of a pentasaccharide-antithrombin complex. We have mutated these two residues to Ala or His to determine their role in the heparin-binding mechanism. The dissociation constants for the binding of both full-length heparin and pentasaccharide to the R46A and R47H variants were increased 3-4-fold and 20-30-fold, respectively, at pH 7.4. Arg46 thus contributes only little to the binding, whereas Arg47 is of appreciable importance. The ionic strength dependence of the dissociation constant for pentasaccharide binding to the R47H variant showed that the decrease in affinity was due to the loss of both one charge interaction and nonionic interactions. Rapid-kinetics studies further revealed that the affinity loss was caused by both a somewhat lower forward rate constant and a greater reverse rate constant of the conformational change step, while the affinity of the initial binding step was unaffected. Arg47 is thus not involved in the initial weak binding of heparin to antithrombin but is important for the heparin-induced conformational change. These results are in agreement with a previously proposed model, in which an initial low-affinity binding of the nonreducing-end trisaccharide of the heparin pentasaccharide induces the antithrombin conformational change. This change positions Arg47 and other residues for optimal interaction with the reducing-end disaccharide, thereby locking the inhibitor in the activated state.     

Conformational changes in serpins: II. The mechanism of activation of antithrombin by heparindagger

Antithrombin, uniquely among plasma serpins acting as proteinase inhibitors in the control of the blood coagulation cascade, circulates in a relatively inactive form. Its activation by heparin, and specifically by a pentasaccharide core of heparin, has been shown to involve release of the peptide loop containing the reactive centre from partial insertion in the A sheet of the molecule. Here we compare the structures of the circulating inactive form of antithrombin with the activated structure in complex with heparin pentasaccharide. We show that the rearrangement of the reactive centre loop that occurs upon activation is part of a widespread conformational change involving a realignment of the two major domains of the molecule. We also examine natural mutants that possess high affinity for heparin pentasaccharide, and relate the kinetics of their interaction with heparin pentasaccharide to the structural transitions occuring in the activation process.