Mechanisms of Glycosaminoglycan-Catalyzed Protease Inactivation by Serpins

丝氨酸蛋白酶抑制剂 (Serpin) 糖胺聚糖催化的蛋白酶灭活机制

• 批准号: 9335436
• 负责人: INGRID M VERHAMME
• 金额: $ 39.38万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9335436
• 关键词: Active Sites; Acylation; Affinity; Agreement; Antithrombins; Atherosclerosis; Behavior; Binding; Binding Sites; Blood Vessels; Catalysis; Chemicals; Circular Dichroism; Clinical; Clinical Research; Complex; Coupled; Crystallization; Crystallography; Data; Dermatan Sulfate; Deuterium; Development; Disease; Docking; Endothelium; Enzymes; Equilibrium; Fluorescence Resonance Energy Transfer; Future; Generations; Glycosaminoglycans; Goals; Hemorrhage; Heparin; Heparin Binding; Heparin Cofactor II; Heparitin Sulfate; Huntington Disease; Hydrogen; Institutes; Kinetics; Ligands; Link; Maryland; Measures; Medical Research; Modeling; Molecular; Molecular Conformation; Mutagenesis; N-terminal; Outcome; Pathology; Pathway interactions; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Plant Roots; Plasma; Play; Positioning Attribute; Process; Property; Proteins; Reaction; Research Personnel; Risk; Role; Serpins; Site; Stents; Structure; System; Tail; Testing; Therapeutic; Thrombin; Time; Tissues; base; design; experience; implantation; in vivo; inhibitor/antagonist; mutant; novel; novel strategies; novel therapeutics; restenosis; therapy design

SUMMARY Extravascular thrombin activity is at the basis of many processes that cause atherosclerosis and in-stent restenosis. The glycosaminoglycans (GAGs) dermatan and heparan sulfate accelerate inactivation of localized thrombin by heparin cofactor II (HCII), but less than 0.1% of the GAGs in vascular tissue is high-affinity heparin that catalyzes thrombin inhibition by tight binding to antithrombin (AT). In agreement with clinical and in-vivo studies, we propose that HCII protects against atherosclerosis and restenosis. The AT mechanism has been analyzed in detail, but the accepted HCII mechanism does not explain its binding and kinetic behavior. Unlike AT, HCII has an intramolecularly sequestered N-terminal tail, thought to be released by GAG binding so it can engage thrombin (T) in the Michaelis complex. HCIIGAG binding is considered to trigger thrombin inactivation by HCII, rather than GAG bridging between thrombin and the serpin, which is at the basis of the AT mechanism. Small GAGs also accelerate thrombin inactivation by HCII but not by AT, which strengthened the assumption that GAG templates play no role in HCII reactions. However, binding of large and small GAGs to HCII is much weaker than to thrombin or AT, implicating a sparsely populated HCII·GAG complex at GAG concentrations that cause maximal inhibition. Inactivation rates parallel T·GAG complex formation, and long GAGs show template kinetics, in disagreement with the accepted mechanism. We aim to define if/how weak HCII·GAG binding can drive catalysis. In the free HCII and T·HCII Michaelis complex structures 70% of the tail is unresolved, and HCIIGAG structures are experimentally unattainable. We will identify tail-body contacts that keep circulating HCII in a low-reactive state, and define structural changes upon GAG binding by hydrogen- deuterium exchange (HDX) MS and circular dichroism, which allow conditions that are prohibitive in crystallography (Aim 1). We will identify for the first time where large GAGs bind across the THCII Michaelis complex, and identify potential binding pockets for small GAGs at the complex interface (Aim 2). We will quantitate the rate steps of GAG binding to HCII and thrombin, and elucidate the kinetic pathways of Michaelis and covalent complex formation by stopped-flow kinetics, equilibrium binding, thrombin inactivation, HCII mutagenesis and FRET (Aim 3). We will test the hypotheses that a) HCII intramolecular tail-body and C sheet- hinge interactions maintain circulating HCII in a low-reactive conformation, activatable to the inhibitory state; b) that T·HCII interface contacts with GAGs stabilize the Michaelis complex with an open-closed equilibrium reflecting exosite I binding of the HCII tail; and c) that the extent of thrombin translocation in the covalent complex may be specific for the HCII-thrombin pair. The expected outcomes will clarify the mechanism of GAG catalysis, and characterize the covalent complex conformation for which no structure is available. The long- term goal is to apply mechanistic information to designing therapies based on HCII and T·HCII-specific GAGs. The findings will be significant for developing novel management of atherosclerosis and restenosis.

概括 血管外凝血酶活性是许多引起动脉粥样硬化和内部的过程的基础 再狭窄。糖胺聚糖（GAGS）皮肤和硫酸乙酰肝素加速局部灭活 肝素辅因子II（HCII）的凝血酶，但血管组织中的GAG少于0.1％是高亲和力肝素 通过紧密结合与抗凝血酶（AT）来催化凝血酶抑制。与临床和体内一致 研究，我们建议HCII预防动脉粥样硬化和再狭窄。 AT机制已经 详细分析了，但是公认的HCII机制不能解释其结合和动力学行为。与众不同 AT，HCII具有分子内隔离的N末端尾巴，被认为是通过插科打结合而释放的，因此可以 参与迈克尔斯建筑群中的凝血酶（T）。 HCIIGAG结合被认为是触发凝血酶失活的 通过HCII，而不是在凝血酶和Serpin之间桥接，这是AT的基础 机制。小插科打s还通过HCII加速了凝血酶灭活，而不是AT，从而加强了凝血酶 假设GAG模板在HCII反应中不起作用。但是，大小插科打结合 HCII比凝血酶或AT弱得多，这暗示了人口稀少的HCII·Gag复合物。 导致最大抑制作用的浓度。灭活率平行t·gag复合物形成，长长 插科打s显示模板动力学，与所接受的机制分歧。我们的目标是定义是否/多么虚弱 HCII插科打结合可以驱动催化。在免费的HCII和T·HCII Michaelis Complex结构中，有70％的尾巴 未解决，HCIIGag结构在实验上是无法实现的。我们将确定尾巴的联系 保持低反应状态的循环HCII，并在氢结合时定义结构变化 氘交换（HDX）MS和圆形二科运动，允许禁止的条件 晶体学（目标1）。我们将首次确定大插口在整个tHciiMichaelis上结合 复合物，并确定在复杂界面处的小插口的潜在结合口袋（AIM 2）。我们将 量化与HCII和凝血酶结合的速率步骤，并阐明Michaelis的动力学途径 通过停止流动动力学，平衡结合，凝血酶失活，HCII，形成共价复合物 诱变和fret（目标3）。我们将测试以下假设：a）HCII分子内尾巴和C板 铰链相互作用在低反应构象中保持循环的HCII，可激活抑制状态。 b） T·HCII接触与插科打的接触使Michaelis Complex稳定了开放式等效br 反映HCII尾巴的外部I结合； c）共价凝血酶易位的程度 复合物可能特定于HCII-thrombin对。预期的结果将阐明堵嘴的机制 催化，并表征没有可用结构的共价复合构象。长期 术语目标是将机械信息应用于基于HCII和T·HCII特异性插科打术的疗法。 这些发现对于发展动脉粥样硬化和再狭窄的新型治疗将很重要。