Residues Tyr253 and Glu255 in strand 3 of β-sheet C of antithrombin are key determinants of an exosite made accessible by heparin activation to promote rapid inhibition of factors Xa and IXa
Residues Tyr253 and Glu255 in strand 3 of β-sheet C of antithrombin are key determinants of an exosite made accessible by heparin activation to promote rapid inhibition of factors Xa and IXa
复制标题
DOI：
10.1074/jbc.m600415200
复制
发表时间：
2006-05-12
影响因子：
4.8
通讯作者：
Olson, ST
中科院分区：
文献类型：
--
作者：
Izaguirre, G;Olson, ST
我们之前的研究表明，肝素对抗凝血丝氨酸（抗凝血酶）的构象激活会在β - sheet C的第3链上产生新的外源位点，从而促进抑制剂与靶蛋白酶Xa因子和IXa因子的反应。为了确定哪些残基包含外源位点，我们突变了在所有脊椎动物抗凝血酶中保守的链3C残基。三个保守的表面可接近残基Tyr（253）、Glu（255）和Lys（257）的组合突变，或仅Tyr（253）和Glu（255）的组合突变，但不是单独的这些残基中的任何一个，都足以在肝素激活的变体与因子Xa和因子IXa的反应中复制链3C抗凝血酶- α(1)-蛋白酶抑制剂嵌合体的外源性缺陷。重要的是，外源性缺陷抗凝血酶与肝素的结合接近野生型亲和力，而肝素激活的突变体与凝血酶的反应接近正常，凝血酶是一种不利用外源性的蛋白酶。保守但部分埋藏的3C链残基Gln(254)，与Glu相互作用的反应性环P6 '残基Arg(399)(255)，或模型研究中提出的构成外源的残基Glu（237）的突变，在缺乏或存在肝素的情况下，都对与凝血酶、因子Xa和因子IXa的抗凝血酶反应性产生极小的影响。综上所述，这些结果表明Tyr（253）和Glu（255）是促进构象激活的抗凝血酶与因子Xa和因子IXa反应的关键外源决定因子。
We previously showed that conformational activation of the anticoagulant serpin, antithrombin, by heparin generates new exosites in strand 3 of beta- sheet C, which promote the reaction of the inhibitor with the target proteases, factor Xa and factor IXa. To determine which residues comprise the exosites, we mutated strand 3C residues that are conserved in all vertebrate antithrombins. Combined mutations of the three conserved surface- accessible residues, Tyr(253), Glu(255), and Lys(257), or of just Tyr(253) and Glu(255), but not any of these residues alone, was sufficient to reproduce the exosite defects of a strand 3C antithrombin- alpha(1)- proteinase inhibitor chimera in reactions of the heparin- activated variants with both factor Xa and factor IXa. Importantly, the exosite- defective antithrombins bound heparin with nearly wild- type affinities, and the heparin- activated mutants showed near normal reactivities with thrombin, a protease that does not utilize the exosite. Mutation of the conserved but partially buried strand 3C residue, Gln(254), the reactive loop P6 ' residue, Arg(399), which interacts with Glu(255), or a residue proposed to constitute the exosite from modeling studies, Glu(237), all produced minimal effects on antithrombin reactivity with thrombin, factor Xa, and factor IXa in the absence or presence of heparin. Together, these results indicate that Tyr(253) and Glu(255) are key exosite determinants responsible for promoting the reactions of conformationally activated antithrombin with both factor Xa and factor IXa.