Mechanism of heparin activation of antithrombin:: Evidence for an induced-fit model of allosteric activation involving two interaction subsites
Mechanism of heparin activation of antithrombin:: Evidence for an induced-fit model of allosteric activation involving two interaction subsites
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DOI：
10.1021/bi981426h
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发表时间：
1998-09-15
期刊：
影响因子：
2.9
通讯作者：
Olson, ST
中科院分区：
文献类型：
--
作者：
Desai, UR;Petitou, M;Olson, ST
肝素五糖DEFGH对丝氨酸蛋白酶抑制剂抗凝血酶的抗凝活化先前显示涉及三糖DEF首先结合并诱导丝氨酸蛋白酶抑制剂的活化，然后是二糖GH结合并稳定活化状态[Petitou等人（1997）Glycobiology 7，323-327; Desai等人（1998）J. Biol，Chem.273，7478-7487]。在本研究中，GH二糖的构象变化和带电残基在变构激活机制中的作用进行了研究，在GH二糖中修饰的变体五糖。通过用-H取代该残基的非必需3-OH来扰动艾杜糖醛酸残基G的构象平衡，导致斜船构象异构体中残基G的分数平行降低（从64%到24%）和五糖与抗凝血酶结合的结合常数[（2.6 +/-0.3）-倍]，与斜船形构象异构体与丝氨酸蛋白酶抑制剂的选择性结合一致。在GH二糖中假定电荷簇侧翼的残基H的3-OH中引入额外的硫酸酯基团，大大增强了对丝氨酸蛋白酶抑制剂的亲和力，增加了35倍，而在斜船构象中残基G的分数仅略有增加（从64%增加到85%）。结合的盐依赖性，连同最近的X-射线结构的抗凝血酶-五糖复合物，表明后者五糖的亲和力增强的大部分是由于直接的静电和氢键的H残基3-O-硫酸与抗凝血酶的相互作用。所有变体戊糖产生抗凝血酶荧光的正常增强和由丝氨酸蛋白酶抑制剂在饱和水平下对因子Xa抑制的正常加速，表明抗凝血酶的构象活化不受戊糖修饰的影响。快速动力学研究与变体五糖的亲和力改变一致，所述亲和力改变主要由还原末端GH二糖与活化的抗凝血酶构象的扰动相互作用引起，并且最小程度地与非还原末端DEF三糖与天然丝氨酸蛋白酶抑制剂构象的结合改变一致。这些结果支持了这样一种模型，其中G残基的构象灵活性促进了向斜舟构象异构体的转化，从而允许GH二糖的带电基团结合并稳定由DEF三糖诱导的活化的抗凝血酶构象。
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 similar to 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.