Abstract
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.
Highlights
Antithrombin is a plasma protein and a member of the serpin family of proteinase inhibitors that can inhibit all of the serine proteinases involved in the blood coagulation cascade [1]
To permit regulation of the location and rate of inhibition of factor Xa, it appears that antithrombin has evolved to require heparin as a high affinity allosteric activator that can shift the position of the equilibrium between ϳ100% A in the absence of heparin to ϳ100% B state upon heparin binding
In this work we examined the role of residues 134 –137, which link helix D to -sheet A, in the heparin activation of antithrombin against factor Xa through transmitting conformational change from the heparin-binding site to the reactive center loop of antithrombin
Summary
Production and Isolation of Variant Forms of Antithrombin—The antithrombin variants were created by site-directed mutagenesis of human antithrombin cDNA on an N135Q background using the Stratagene Quik Change Kit, following the manufacturer’s directions. Because of the decreased fluorescence enhancement upon heparin binding seen for the antithrombin deletion variants, tryptophan fluorescence, which is normally exploited to determine heparin affinity [15], could not be used to obtain accurate heparin affinities. Heparin binding to antithrombin-TNS causes a change in the environment of the antithrombin-bound TNS, resulting in an approximately 50 – 60% decrease in TNS fluorescence intensity and a blue shift of the emission maximum. To determine the antithrombin-heparin dissociation constants for the variants, antithrombin in the presence of a 20-fold excess of TNS was titrated with heparin. For control antithrombin and the ⌬137 variant, 250 nM antithrombin and 5 M TNS were used for both pentasaccharide and full-length heparin titrations. For the rest of the deletion variants, 250 or 300 nM antithrombin and 5 or 6 M TNS were used for full-length heparin titrations, and 250 –500.
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