Abstract
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.
Highlights
Antithrombin, a member of the serpin family of protease inhibitors [1,2,3,4], is a key regulator of blood coagulation
We examined the effect of a proline substitution at Lys133 in the extended heparin-binding region and found that a blocking or distortion of the extension of helix D resulted in the inability to become fully activated, as measured by fluorescence enhancement and kinetics of inhibition
Rapid heparin binding studies indicated that the reason full activation was not achieved is that the conformational changes that stabilize the high affinity state were perturbed
Summary
Antithrombin, a member of the serpin family of protease inhibitors [1,2,3,4], is a key regulator of blood coagulation. In 1994, based solely on the structure of native antithrombin and molecular dynamics simulations, van Boeckel et al [13] proposed a mechanism whereby charge neutralization at the heparin binding region would cause helix D elongation, resulting in strain on strand 2A and the collapse of -sheet A to the five-stranded form [4]. This model was largely validated by the structure of the pentasaccharide-activated antithrombin. We demonstrate that helix D elongation is coupled to the activating conformational change and is crucial for trapping the equilibrium in the activated state
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