STRUCTURAL STUDIES ON SERINE PROTEASES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这项建议侧重于与生物相关的丝氨酸蛋白酶与我们目前缺乏结构信息的抑制剂、底物和效应物形成的复合体。重点是凝血酶和组织型纤溶酶原激活物，这两种参与血栓形成和溶解的关键酶，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，使其在体内具有最佳的抗凝活性，或者在底物水解性方面存在缺陷。制备了W215残基的一系列突变体，并与活性位点抑制剂PPACK和PPPCK形成复合体，以探索与酶的S3位点和初级专一性口袋的相互作用方式。随着突变体W215/E217A准备进入临床试验，双突变体W215/E217A的晶体将揭示其非凡体内效力的分子基础。凝血酶S195A和D102N的非活性形式在不同的结晶阶段与蛋白酶激活的受体PAR1、PAR3和PAR4的片段、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形成复合体。这些复合体的结构将使我们对血液中凝血酶促凝剂、血栓形成和抗凝血活性的分子基础的了解取得重大进展。凝血酶突变体也将在没有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已经以自由形式结晶，我们渴望寻找更高分辨率的结构来验证先前结构研究中未能获得的生物相关30-环的构象。TPA还将与生理抑制剂PAI-1形成复合体，以确定识别表位并促进药物干预。

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.