Conformational change propagation in ATIII-heparin

描述（由申请人提供）：必需抗凝血蛋白抗凝血酶III （ATIII）使用蛇形蛋白自杀底物机制与凝血酶形成抑制复合物。然而，天然循环ATIII是一种低效的蛋白酶抑制剂，因为它的反应环部分插入其中央A -片。为了完全激活，辅助因子肝素必须结合ATIII并诱导蛋白质构象改变，从而导致反应性环排出和fXa抑制率增加约300倍。肝素激活ATIII的机制在辅助因子与抑制剂结合的第一阶段和反应性环排出的最后阶段得到了很好的理解。相比之下，对于负责从五糖结合位点到反应环的激活构象变化传播的中间步骤所知甚少。这项工作的目标是确定介导构象变化传递的ATIII结构元件和相互作用，并更好地了解这种重要的药学变构激活机制。我们的工作模型提出，从五糖- atiii界面辐射出3个构象变化。Prong-1导致螺旋P （hP）的形成，然后与prong-2（通过n端多肽传递）汇合，重组hE臂并旋转Y166。Y166旋转促进了抑制剂赤道处的薄片- a闭合。在反应环极的A片闭合同样被3叉介导的Y131旋转促进。（在ATIII与五糖非还原端结合后，探针3通过hA、sB和hD传递。）当a片的2链和3链离开hD和hE时，它们从hF臂下通过，hF臂与a片残基网络直接稳定插入的反应环和部分打开的天然构象a片。酪氨酸旋转驱动的s2A/s3A在hF臂下的运动破坏了这个天然网络，并释放其组分参与不同的连接，以稳定p14排出的封闭A-sheet活化分子。通过破坏假设的、关键的结构相互作用，并确定这些变化如何影响肝素亲和力和结合动力学、fXa抑制的变构激活和突变体的分子结构，将评估和完善ATIII中肝素依赖的构象变化传播模型。

DESCRIPTION (provided by applicant): The essential anticoagulant protein antithrombin III (ATIII) uses a serpin suicide substrate mechanism to form inhibitory complexes with clotting enzymes. However, native circulating ATIII is an inefficient proteinase inhibitor due to partial insertion of its reactive loop in its central A beta-sheet. For full activation, cofactor heparin must bind ATIII and induce a protein conformational change that leads to reactive loop expulsion and about 300x increase in the fXa inhibition rate. The mechanism of ATIII activation by heparin is reasonably well understood at the first stage of cofactor binding to the inhibitor and the final stage of reactive loop expulsion. In contrast, much less is known about intermediate steps responsible for propagation of the activating conformational change from the pentasaccharide-binding site to the reactive loop. The goals of this work are to identify ATIII structural elements and interactions that mediate conformational change transmission, and to develop a better understanding of this pharmaceutically important allosteric activation mechanism. Our working model proposes that 3 prongs of conformational change radiate from the pentasaccharide-ATIII interface. Prong-1 leads to helix P (hP) formation and then converges with prong-2 (which is transmitted through the N-terminal polypeptide) to restructure the hE arm and rotate Y166. Y166 rotation promotes sheet-A closure at the equator of the inhibitor. Sheet A closure at the reactive loop pole is similarly promoted by prong-3-mediated Y131 rotation. (Prong 3 is relayed through hA, sB and hD following ATIII binding to the pentasaccharide non-reducing end.) As strands 2 and 3 of sheet-A move away from hD and hE, they pass under the hF arm which with a network of A-sheet residues directly stabilizes the inserted reactive loop and partially opened A-sheet of the native conformation. Tyrosine rotation-driven movement of s2A/s3A under the hF arm disrupts this native network and frees its components to engage in different linkages that stabilize the P14-expelled closed A-sheet activated molecule. The model for heparin dependent conformational change propagation in ATIII will be evaluated and refined by disrupting hypothesized, critical structural interactions and determining how such changes affect heparin affinity and binding kinetics, allosteric activation of fXa inhibition and the molecular structures of the mutants.