This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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.
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