STRUCTURAL STUDIES ON SERINE PROTEASES

丝氨酸蛋白酶的结构研究

• 批准号: 7956836
• 负责人: Enrico Di Cera
• 金额: $ 2.83万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7956836
• 关键词: Active Sites; Alteplase; Anticoagulants; Blood; Blood Clot; Blood coagulation; Cations; Clinical Trials; Complex; Computer Retrieval of Information on Scientific Projects Database; Crystallization; Engineering; Enzymes; Epitopes; F2R gene; Factor V; Factor VIII; Funding; Grant; Hydrolysis; Institution; Intervention; Ligands; Molecular; Molecular Conformation; Monovalent Cations; PAWR gene; Peptide Hydrolases; Physiological; Plasminogen Activator Inhibitor 1; Protein C; Proteinase-Activated Receptors; Research; Research Personnel; Resolution; Resources; Series; Serine Protease; Site; Source; Specificity; Staging; Structure; Thrombin; Thrombomodulin; United States National Institutes of Health; base; in vivo; inhibitor/antagonist; mutant; phenylalanyl-prolyl-arginine-chloromethyl ketone; receptor; structural biology

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This proposal focuses on biologically relevant serine proteases in complex with inhibitors, substrates and effectors for which we currently lack structural information. The focus is mainly on thrombin and tissue-type plasminogen activator, two key proteases involved in the formation and dissolution of blood clots and major targets of pharmacological intervention. We plan to crystallize several thrombin mutants engineered for optimal anticoagulant activity in vivo, or to be defective for substrate hydrolysis. A series of mutants of residue W215 have been prepared and crystallized in complex with active site inhibitors PPACK and PPPCK to probe the mode of interaction with the S3 site and primary specificity pocket of the enzyme. Crystals of the double mutant W215/E217A will reveal the molecular basis of its remarkable in vivo potency as the mutant prepares to enter clinical trials. Inactive forms of thrombin S195A and D102N are in various stages of crystallization in complex with fragments of the protease activated receptors PAR1, PAR3 and PAR4, the receptor thrombomodulin, and physiological substrates like factor V, factor VIII and protein C. Structures of these complexes will produce major advances in our understanding of the moelcular basis of thrombin procoagulant, prothrombotic and anticoagulant activities in the blood. Thrombin mutants will also be crystallized in the absence of ligands and in the presence of different monovalent cations to determine the basis of cation specificity. tPA has been crystallized in the free form and we are eager to pursue a higher resolution structure to verify the conformation of the biologically relevant 30-loop that has eluded previous structural studies. tPA will also be crystallized in complex with the physiological inhibitor PAI-1 to determine the epitopes of recognition and to facilitate pharmacological intervention.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 该提案重点关注与抑制剂、底物和效应物复合的生物学相关丝氨酸蛋白酶，目前我们缺乏这些丝氨酸蛋白酶的结构信息。重点关注凝血酶和组织型纤溶酶原激活剂，这两种参与血栓形成和溶解的关键蛋白酶，也是药物干预的主要靶点。我们计划结晶几种凝血酶突变体，这些突变体被设计为具有最佳的体内抗凝血活性，或者具有底物水解缺陷。已制备了一系列残基 W215 突变体，并与活性位点抑制剂 PPACK 和 PPPCK 形成复合物结晶，以探索与酶的 S3 位点和初级特异性口袋的相互作用模式。当突变体准备进入临床试验时，双突变体 W215/E217A 的晶体将揭示其显着体内效力的分子基础。非活性形式的凝血酶 S195A 和 D102N 与蛋白酶激活受体 PAR1、PAR3 和 PAR4、受体血栓调节蛋白以及生理底物（如因子 V、因子 VIII 和蛋白 C）的片段形成复合物，处于不同的结晶阶段。这些复合物的结构将在我们对凝血酶促凝、促血栓和抗凝活性的分子基础的理解方面取得了重大进展 血。凝血酶突变体也将在不存在配体和存在不同单价阳离子的情况下结晶，以确定阳离子特异性的基础。 tPA 已以游离形式结晶，我们渴望寻求更高分辨率的结构，以验证生物学相关的 30 环的构象，而这在之前的结构研究中是无法解决的。 tPA 还将与生理抑制剂 PAI-1 形成复合物结晶，以确定识别表位并促进药理干预。