STRUCTURAL STUDIES ON SERINE PROTEASES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 该提案的重点是生物学相关的丝氨酸蛋白酶与抑制剂，底物和效应物的复合物，我们目前缺乏结构信息。重点主要是凝血酶和组织型纤溶酶原激活剂，这两种关键蛋白酶参与血凝块的形成和溶解，也是药物干预的主要靶点。我们计划结晶几个凝血酶突变体工程最佳抗凝活性在体内，或有缺陷的底物水解。已制备了一系列残基W215的突变体，并与活性位点抑制剂PPACK和PPPCK复合结晶，以探测与酶的S3位点和主要特异性口袋的相互作用模式。双突变体W215/E217 A的晶体将揭示其显着的体内效力的分子基础，因为突变体准备进入临床试验。凝血酶S195 A和D102 N的非活性形式处于与蛋白酶激活的受体PAR 1、PAR 3和PAR 4的片段、受体血栓调节蛋白和生理底物如因子V、因子VIII和蛋白C复合的不同结晶阶段。这些复合物的结构将产生重大进展，在我们的理解凝血酶促凝血，促血栓形成和抗凝血活动的分子基础。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已经以游离形式结晶，我们渴望追求更高分辨率的结构，以验证生物学相关的30-环的构象，这在以前的结构研究中是无法实现的。tPA也将与生理抑制剂派-1复合结晶，以确定识别表位并促进药理学干预。

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.