Abstract

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. The variants exhibited basal reactivities with factors Xa and IXa, heparin affinities and thermal stabilities that were dramatically altered from wild type, consistent with the P14 mutations perturbing native state stability and shifting an allosteric equilibrium between native and activated states. Rapid kinetic studies confirmed that limiting rate constants for heparin allosteric activation of the mutants were altered in conjunction with the observed shifts of the allosteric equilibrium. However, correlations of the P14 mutations' effects on parameters reflecting the allosteric activation state of the serpin were inconsistent with a two-state model of allosteric activation and suggested multiple activated states. Together, these findings support a minimal three-state model of allosteric activation in which the P14 mutations perturb equilibria involving distinct native, intermediate, and fully activated states wherein the P14 residue retains an interaction with the hydrophobic core in the intermediate state but is released from the core in the fully activated state, and the bulk of allosteric activation has occurred in the intermediate.

Highlights

  • Structural changes in the antithrombin reactive center loop P14 residue are thought to mediate heparin allosteric activation

  • Correction of ka, obs in the latter plot for a limiting ka, N of 1500 MϪ1 sϪ1 resulted in linearization of this plot. These results suggested that changes in heparin affinity of the mutants paralleled changes in basal reactivity with factors Xa and IXa, but this correlation deviated from that expected for a two-state allosteric equilibrium, i.e. the fraction of activated antithrombin predicted by the two parameters did not correspond

  • Rapid Kinetics of Heparin Binding to Antithrombin Variants—To determine whether the P14 residue mutations affected heparin affinity for antithrombin by perturbing the allosteric equilibrium between native and activated states, we investigated the kinetics of heparin pentasaccharide binding to the variant antithrombins by monitoring fluorescence changes reporting binding under pseudo-first order conditions

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Summary

Introduction

Structural changes in the antithrombin reactive center loop P14 residue are thought to mediate heparin allosteric activation. 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. Correlations of the P14 mutations’ effects on parameters reflecting the allosteric activation state of the serpin were inconsistent with a two-state model of allosteric activation and suggested multiple activated states Together, these findings support a minimal three-state model of allosteric activation in which the P14 mutations perturb equilibria involving distinct native, intermediate, and fully activated states wherein the P14 residue retains an interaction with the hydrophobic core in the intermediate state but is released from the core in the fully activated state, and the bulk of allosteric activation has occurred in the intermediate

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