Desai U R, Petitou M, Björk I, Olson S T
Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago 60612, USA.
Biochemistry. 1998 Sep 15;37(37):13033-41. doi: 10.1021/bi981426h.
The anticoagulant activation of the serpin antithrombin by heparin pentasaccharide DEFGH was previously shown to involve trisaccharide DEF first binding and inducing activation of the serpin, followed by disaccharide 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 activation 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 of 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 association constant for pentasaccharide binding to antithrombin [(2.6 +/- 0.3)-fold], consistent with selective binding of the skew boat conformer to the serpin. Introduction of an additional sulfate group to the 3-OH of residue H flanking a putative charge cluster in the GH disaccharide greatly enhanced the affinity for the serpin by approximately 35-fold with only a small increase in the fraction of residue G in the skew boat conformation (from 64 to 85%). The salt dependence of binding, together with a recent X-ray structure of the antithrombin-pentasaccharide complex, suggested that the majority of the enhanced affinity of the latter pentasaccharide was due to direct electrostatic and hydrogen-bonding interactions of the H residue 3-O-sulfate with antithrombin. All variant pentasaccharides produced a normal enhancement of antithrombin fluoresence and normal acceleration of factor Xa inhibition by the serpin at saturating levels, indicating that conformational activation of antithrombin was not affected by the pentasaccharide modifications. Rapid kinetic studies were consistent with the altered affinities of the variant pentasaccharides resulting mostly from perturbed interactions of the reducing-end GH disaccharide with the activated antithrombin conformation and minimally to an altered binding of the nonreducing-end DEF trisaccharide to the native serpin conformation. Together, these results support a model in which the conformational flexibility 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 the DEF trisaccharide.
先前的研究表明，肝素五糖DEFGH对丝氨酸蛋白酶抑制剂抗凝血酶的抗凝激活作用，首先是三糖DEF结合并诱导丝氨酸蛋白酶抑制剂激活，随后二糖GH结合并稳定激活状态[佩蒂图等人（1997年）《糖生物学》7，323 - 327；德赛等人（1998年）《生物化学杂志》273，7478 - 7487]。在本研究中，通过对GH二糖进行修饰的变体五糖，研究了GH二糖的构象变化和带电残基在变构激活机制中的作用。通过将艾杜糖醛酸残基G的非必需3 - OH用 - H取代，扰动该残基的构象平衡，导致残基G在扭船构象中的比例平行下降（从64%降至24%），以及五糖与抗凝血酶结合的缔合常数下降[（2.6 ± 0.3）倍]，这与扭船构象与丝氨酸蛋白酶抑制剂的选择性结合一致。在GH二糖中一个假定的电荷簇侧翼的残基H的3 - OH上引入一个额外的硫酸基团，极大地增强了对丝氨酸蛋白酶抑制剂的亲和力，约为35倍，而残基G在扭船构象中的比例仅略有增加（从64%增至85%）。结合的盐依赖性以及抗凝血酶 - 五糖复合物最近的X射线结构表明，后一种五糖增强的亲和力主要是由于残基H的3 - O - 硫酸与抗凝血酶的直接静电和氢键相互作用。所有变体五糖在饱和水平下均使抗凝血酶荧光正常增强，并且使丝氨酸蛋白酶抑制剂对因子Xa的抑制正常加速，表明抗凝血酶的构象激活不受五糖修饰的影响。快速动力学研究表明，变体五糖亲和力的改变主要源于还原端GH二糖与激活的抗凝血酶构象相互作用的扰动，而对非还原端DEF三糖与天然丝氨酸蛋白酶抑制剂构象结合改变的影响最小。总之，这些结果支持了一个模型，即残基G的构象灵活性促进了向扭船构象的转变，从而使GH二糖的带电基团能够结合并稳定由DEF三糖诱导的激活的抗凝血酶构象。
