Conformational change propagation in ATIII-heparin

描述（由申请方提供）：必需抗凝蛋白抗凝血酶III（ATIII）使用丝氨酸蛋白酶抑制剂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，因为其反应环部分插入其中心A β折叠中。对于完全激活，辅因子肝素必须结合ATIII并诱导蛋白质构象变化，导致反应性环排出和fXa抑制率增加约300倍。在辅因子结合抑制剂的第一阶段和反应环排出的最后阶段，肝素激活ATIII的机制得到了合理的理解。相比之下，少得多的是已知的中间步骤负责传播的激活构象变化从pentasglide结合位点的反应环。这项工作的目标是确定ATIII结构元件和介导构象变化传递的相互作用，并更好地了解这种药学上重要的变构激活机制。我们的工作模型提出，3个叉的构象变化辐射从pentassistide-ATIII接口。叉-1导致螺旋P（hP）形成，然后与叉-2（其通过N-末端多肽传递）会聚以重构hE臂并旋转Y166。Y166旋转促进片材-A在抑制剂的赤道处闭合。类似地，prong-3介导的Y131旋转也促进了反应回路极点处的片A闭合。（在ATIII结合至五糖非还原末端之后，叉头3通过hA、sB和hD中继。）当A片层的链2和3远离hD和hE移动时，它们在hF臂下通过，该hF臂与A片层残基的网络一起直接稳定插入的反应性环和天然构象的部分打开的A片层。酪氨酸旋转驱动的s2 A/s3 A在hF臂下的运动破坏了这种天然网络，并释放其组分以参与不同的连接，这些连接稳定了P14排出的闭合A折叠活化分子。ATIII中肝素依赖性构象变化传播的模型将通过破坏假设的关键结构相互作用并确定此类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来进行评价和完善。

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.