MECHANISM OF HEPARIN ACTIVATION OF ANTITHROMBIN - EVIDENCE FOR AN INDUCED-FIT MODEL OF ALLOSTERIC ACTIVATION INVOLVING 2 INTERACTION SUBSITES

The anticoagulant activation of the serpin antithrombin by heparin pen tasaccharide DEFGH was previously shown to involve trisaccharide DEF f irst binding and inducing activation of the serpin, followed by disacc haride GH binding and stabilizing the activated state [Petitou et al. (1997) Glycobiology 7, 323-327; Desai et al. (1998) J. Biol, Chem. 273 , 7478-7487]. In the present study, the role of conformational changes and charged residues of the GH disaccharide in the allosteric activat ion mechanism was investigated with variant pentasaccharides modified in the GH disaccharide. Perturbation of the conformational equilibrium of iduronate residue G through replacement of the nonessential 3-OH o f this residue with -H resulted in parallel decreases in the fraction of residue G in the skew boat conformer (from 64 to 24%) and in the as sociation constant for pentasaccharide binding to antithrombin [(2.6 /- 0.3)-fold], consistent with selective binding of the skew boat conf ormer to the serpin. Introduction of an additional sulfate group to th e 3-OH of residue H flanking a putative charge cluster in the GH disac charide greatly enhanced the affinity for the serpin by similar to 35- fold with only a small increase in the fraction of residue G in the sk ew boat conformation ( from 64 to 85%). The salt dependence of binding , together with a recent X-ray structure of the antithrombin-pentasacc haride complex, suggested that the majority of the enhanced affinity o f the latter pentasaccharide was due to direct electrostatic and hydro gen-bonding interactions of the H residue 3-O-sulfate with antithrombi n. All variant pentasaccharides produced a normal enhancement of antit hrombin fluoresence and normal acceleration of factor Xa inhibition by the serpin at saturating levels, indicating that conformational activ ation of antithrombin was not affected by the pentasaccharide modifica tions. Rapid kinetic studies were, consistent with the altered affinit ies of the variant pentasaccharides resulting mostly from perturbed in teractions of the reducing-end GH disaccharide with the activated anti thrombin conformation and minimally to an altered binding of the nonre ducing-end DEF trisaccharide to the native serpin conformation, Togeth er, these results support a model in which the conformational flexibil ity of the G residue facilitates conversion to the skew boat conformer and thereby allows charged groups of the GH disaccharide to bind and stabilize the activated antithrombin conformation that is induced by t he DEF trisaccharide.