STRUCTURES OF CATALYTIC DOMAINS OF BLOOD PROTEINS

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The immediate objective of this work is to solve and refine the structure of human PPACK-alpha-thrombin. The structure of this prototype of serine protease catalytic domains will reveal the manner in which the highly selective fibrinogen-thrombin interaction is accomplished. Moreover, since many of the enzyme domains of blood coagulation/fibrinolysis are highly homologous, the human alpha-thrombin structure will be used to model closely homologous regions of these other proteases as well as model differences between species. The structure of alpha-thrombin will then be used in molecular replacement application to solve the structures of the hirudin complexes of alpha- and PPACK-alpha-thrombin. These studies are designed to localize the binding sites of the anti-thrombotic hirudin and the thrombin molecules to delineate the three dimensional and other factors responsible for the incredibly singular binding affinity between these molecules (femto and pico molar, respectively). Similar methods will be employed to determine the structures of prothrombin 1 and single chain prothrombin 2. In the case of the former, rotation-translation searches will also include the three disulfide, triple loop of the kringle 2- catalytic domain interactions of prothrombin; a comparison of the pre- thrombin 2 and thrombin structures will reveal the conformational changes occurring upon activation cleavage of prothrombin by Factor Xa. A longer range thrust of the work will center upon the structure determination of intact cassette domain molecules such as the thrombolytics tissue plasminogen activator (TPA) and prourokinase. The scope of the effort will be broadened and enhanced by also attempting the crystallization of third generation, deglycosylated exon shuffled constructs of the TPA molecule. The number of possibilities of interchanging and/or deleting finger, growth factor, kringle and catalytic domains is attractively large thus favorably increasing the probability of uncovering crystallizable constructs.