Conformational change propagation in ATIII-heparin

ATIII-肝素中的构象变化传播

• 批准号: 7331505
• 负责人: SUSAN C BOCK
• 金额: $ 31.89万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7331505
• 关键词: Affect; Affinity; Anticoagulants; Antithrombin III; Antithrombins; Binding; Binding Sites; Closure; Complex; Elements; Event; Foundations; Goals; Helix (Snails); Heparin; Kinetics; Mediating; Modeling; Molecular Conformation; Molecular Structure; Movement; N-terminal; Protease Inhibitor; Proteins; Rate; Rotation; Series; Serpins; Staging; Tyrosine; Upper arm; Work; beta pleated sheet; clotting enzyme; cofactor; ear helix; heparin cofactor; inhibitor/antagonist; mutant; polypeptide; suicide substrates; transmission process

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.

描述（由申请人提供）：必需的抗凝血蛋白抗凝血酶 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央 A β 片层。为了完全激活，辅因子肝素必须结合 ATIII 并诱导蛋白质构象变化，从而导致反应环排出并使 fXa 抑制率增加约 300 倍。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，肝素激活 ATIII 的机制已得到相当充分的了解。相比之下，我们对负责激活构象变化从五糖结合位点传播到反应环的中间步骤知之甚少。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，并更好地理解这种药学上重要的变构激活机制。我们的工作模型提出构象变化的三个分支从五糖-ATIII 界面辐射出来。 Prong-1 导致螺旋 P (hP) 形成，然后与 Prong-2（通过 N 端多肽传递）会聚，重组 hE 臂并旋转 Y166。 Y166 旋转促进片材 A 在抑制剂赤道处闭合。类似地，prong-3 介导的 Y131 旋转也会促进反应环极处的片材 A 闭合。 （在 ATIII 与五糖非还原端结合后，分支 3 通过 hA、sB 和 hD 中继。）当片状 A 的第 2 条和第 3 条链从 hD 和 hE 移开时，它们在 hF 臂下方通过，该臂具有以下网络： A-片残基直接稳定插入的反应环和部分打开的天然构象的A-片。 hF 臂下酪氨酸旋转驱动的 s2A/s3A 运动破坏了这个天然网络，并释放其组件以参与不同的连接，从而稳定 P14 排出的闭合 A 片层激活分子。 ATIII 中肝素依赖性构象变化传播模型将通过破坏假设的关键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 抑制的变构激活以及突变体的分子结构来评估和完善。