IDENTIFICATION OF THE ANTITHROMBIN-III HEPARIN-BINDING SITE

The heparin binding site of the anticoagulant protein antithrombin III (ATIII) has been defined at high resolution by alanine scanning mutag enesis of 17 basic residues previously thought to interact with the co factor based on chemical modification experiments, analysis of natural ly occurring dysfunctional antithrombins, and proximity to helix D. Th e baculovirus expression system employed for this study produces antit hrombin which is highly similar to plasma ATIII in its inhibition of t hrombin and factor Xa and which resembles the naturally occurring beta -ATIII isoform in its interactions with high affinity heparin and pent asaccharide (Ersdal-Badju, E., Lu, A., Peng, X., Picard, V., Zendehrou h, P., Turk, B., Bjork, I., Olson, S. T., and Beck, S. C. (1995) Bioch em. J. 310, 323-330). Relative heparin affinities of basic-to-Ala subs titution mutants were determined by NaCl gradient elution from heparin columns. The data show that only a subset of the previously implicate d basic residues are critical for binding to heparin. The key heparin binding residues, Lys-ll, Arg-13, Arg-24, Arg-47, Lys-125, Arg-129, an d Arg-145, line a 50-Angstrom long channel on the surface of ATIII. Co mparisons of binding residue positions in the structure of P14-inserte d ATIII and models of native antithrombin, derived from the structures of native ovalbumin and native antichymotrypsin, suggest that heparin may activate antithrombin by breaking salt bridges that stabilize its native conformation. Specifically, heparin release of intramolecular helix D-sheet B salt bridges may facilitate s123AhDEF movement and gen eration of an activated species that is conformationally primed for re active loop uptake by central beta-sheet A and for inhibitory complex formation. In addition to providing a structural explanation for the c onformational change observed upon heparin binding to antithrombin III , differences in the affinities of native, heparin-bound, complexed, a nd cleaved ATIII molecules for heparin can be explained based on the i dentified binding site and suggest why heparin functions catalytically and is released from antithrombin upon inhibitory complex formation.