STRUCTURAL STUDIES ON SERINE PROTEASES

丝氨酸蛋白酶的结构研究

• 批准号: 7725986
• 负责人: Enrico Di Cera
• 金额: $ 3.17万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7725986
• 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; Endopeptidases; 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

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 该提案着重于与抑制剂，底物和效应子中我们目前缺乏结构信息的复杂物相关的丝氨酸蛋白酶。重点主要放在凝血酶和组织型纤溶酶原激活剂上，这是两个关键蛋白酶，涉及血块的形成和溶解以及药理干预的主要靶标。我们计划结晶几个用于体内最佳抗凝活性的凝血酶突变体，或者在底物水解中有缺陷。已经制备了一系列残基W215的突变体，并与活性位点抑制剂PPACK和PPPCK结晶，以探测与S3位点的相互作用方式以及酶的主要特异性袋。双突变体W215/E217A的晶体将揭示其在体内效力显着的分子基础，因为该突变体准备进入临床试验。凝血酶S195A和D102N的不活性形式在复合体的各个阶段与蛋白酶活化受体PAR1，PAR3和PAR4的碎片，受体血栓瘤蛋白，生理底物以及因子V，因子VIII和蛋白质C等生理底物在这些复合物中产生主要的培养基，使我们的理解能够产生主要的发展。血液中的抗凝活性。在不存在配体的情况下和存在不同的单价阳离子的情况下，凝血酶突变体也将结晶，以确定阳离子特异性的基础。 TPA已以自由形式结晶，我们渴望追求更高的分辨率结构，以验证已经避免了先前结构研究的生物学相关30循环的构象。 TPA还将与生理抑制剂PAI-1结晶，以确定识别表位并促进药理干预。