Conformational change propagation in ATIII-heparin

描述(由申请人提供)：基本抗凝血蛋白抗凝血酶III(ATIII)使用丝氨酸自杀底物机制与凝血酶形成抑制复合体。然而，天然循环的ATIII是一种低效的蛋白酶抑制剂，因为它的反应环部分插入到其中心Aβ-折叠中。为了充分激活，辅因子肝素必须与ATIII结合，并诱导蛋白质构象变化，导致反应性环排出，FXA抑制率增加约300倍。肝素激活ATIII的机制在辅因子与抑制剂结合的第一阶段和反应环排出的最后阶段被很好地理解。相反，对于负责将激活的构象变化从五糖结合部位传播到反应环的中间步骤，我们知道的要少得多。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，并更好地了解这一具有重要药学意义的变构激活机制。我们的工作模型提出了三种构象变化从五糖-ATIII界面辐射出来。Prong-1导致螺旋P(HP)的形成，然后与Prong-2(通过N-末端多肽传递)聚合，重组He臂并旋转Y166。Y166旋转促进了缓蚀剂赤道处的片状A闭合。Pong-3介导的Y131转动类似地促进了反应环极点的A板闭合。(在ATIII与五糖非还原末端结合后，Pong 3通过HA、SB和HD传递。)当Sheet-A的2和3链离开HD和He时，它们在HF臂下通过，HF臂与A-Sheet残基网络一起直接稳定插入的反应环和部分打开的天然构象的A-Sheet。S2A/S3A在HF臂下的酪氨酸旋转驱动的运动破坏了这一天然网络，并使其成分自由地参与不同的连接，从而稳定了P14-排出的闭合A-折叠激活分子。通过破坏假设的关键结构相互作用，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，将对ATIII中肝素依赖的构象变化传播模型进行评估和改进。

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.