Disruption of a tight cluster surrounding tyrosine 131 in the native conformation of antithrombin III activates it for factor Xa inhibition
Disruption of a tight cluster surrounding tyrosine 131 in the native conformation of antithrombin III activates it for factor Xa inhibition
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DOI：
10.1074/jbc.m604826200
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发表时间：
2006-10-20
影响因子：
4.8
通讯作者：
Bock, Susan C.
中科院分区：
文献类型：
--
作者：
dela Cruz, Richard Glenn C.;Jairajpuri, Mohamad Aman;Bock, Susan C.
抗凝血酶III（ATIII）的天然构象是其凝血途径靶酶的不良抑制剂，因为其反应中心环（RCL）部分插入其中心A β折叠中。这项研究集中在酪氨酸131，这是位于螺旋D-片层A界面，毗邻ATIII五糖和肝素辅因子结合位点和一些17 A远离RCL插入。晶体结构表明，Tyr（131）环隐藏在天然ATIII中，当五糖与抑制剂结合并激活抑制剂时，Tyr（131）环暴露出来，这表明Tyr（131）可能是ATIII构象激活的开关。该假设得到了本研究结果的支持，该研究逐渐从Tyr（131）侧链上去除原子。肝素非依赖性Y131 L和Y131 A因子Xa抑制速率分别比对照和Y131 F快25倍和29倍，表明Tyr（131）环与相邻螺旋D和链2A残基的相互作用将未催化的天然-活化构象平衡向RCL插入状态转移。热变性实验表明，Y131 A和Y131 L的稳定性低于对照和Y131 F，这意味着在这些遗传活化的分子中A-折叠迁移率的趋势增加。因此，天然抗凝血酶的螺旋D-链2A界面处的紧密Tyr（131）-Asn（127）-Leu（130）-Leu（140）-Ser（142）簇对基态构象的稳定性有显著贡献，并且酪氨酸131在ATIII抗凝活性的变构激活期间充当肝素响应性分子开关。
The native conformation of antithrombin III (ATIII) is a poor inhibitor of its coagulation pathway target enzymes because of the partial insertion of its reactive center loop (RCL) in its central A beta-sheet. This study focused on tyrosine 131, which is located at the helix D-sheet A interface, adjacent to the ATIII pentasaccharide and heparin cofactor-binding sites and some 17 A away from the RCL insertion. Crystallographic structures show that the Tyr(131) ring is buried in native ATIII and then becomes exposed when pentasaccharide binds to the inhibitor and activates it. This change suggested that Tyr(131) might serve as a switch for ATIII conformational activation. The hypothesis is supported by results from this study, which progressively removed atoms from the Tyr(131) side chain. Rates of heparin-independent Y131L and Y131A factor Xa inhibition were 25 and 29 times faster than for the control and Y131F, suggesting that Tyr(131) ring interactions with neighboring helix D and strand 2A residues shift the uncatalyzed native-to-activated conformational equilibrium toward the RCL-inserted state. Thermal denaturation experiments showed Y131A and Y131L were less stable than the control and Y131F, implying an increased tendency toward A-sheet mobility in these genetically activated molecules. Thus, the tight Tyr(131)-Asn(127)-Leu(130)-Leu(140)-Ser(142) cluster at the helix D-strand 2A interface of native antithrombin contributes significantly to the stability of the ground state conformation, and tyrosine 131 serves as a heparin-responsive molecular switch during the allosteric activation of ATIII anticoagulant activity.