CRYSTALLOGRAPHIC STRUCTURES OF KRINGLES AND GLA-DOMAINS

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The long range objectives of this work are to place some of the events of the blood coagulation cascade on a three dimensional structural basis and to do likewise for aspects of fibrinolysis; the very processes of life are directly dependent upon the proper execution of both of these functions. The goals are to be achieved by determining the crystallographic structures of a number of molecules involved in the processes and then extending the information to a much larger group of closely interrelated molecules by computer graphics modeling with crystallographic coordinates, high field NMR observations, electrostatic calculations and energy minimization refinements. We have already demonstrated the feasibility of this approach by modeling he Lys subsite of the fibrin binding site of several fibrin binding kringle structures, including tissue plasminogen activator (TPA), based on the structure of prothrombin fragment 1. We plan to expand the observed kringle structure library with the structure determination of K4 of plasminogen and prothrombin fragment 2. Thereafter, we will model the remaining eight kringles of five different blood proteins and the 37 highly conserved K4 kringles of apolipoprotein(a). With an expanded kringle library, kringle- kringle packing interactions in multiple kringle proteins will be approximated based on packing in four different crystal structures, computer graphics and the structure of fragment 1-2. The Gla-domain of prothrombin occurs in six other proteins of coagulation and along with Ca+2 is responsible for membrane binding of these molecules. The macromolecular membrane complexes enhance catalysis in a spectacular way. We are on the verge of solving the structure of Ca+2-fragment 1 which has undergone the conformational transition demanded of membrane binding. The structure will be used to model the other Gla-domains that show even greater conservation than kringles. The Gla-Ca+2-phospholipid interaction will be fixed from crystallographic binding studies with small organic phosphates and the dynamics of the conformational changed will be approached by determining the structure of Mg+2-fragment 1 which displays a different conformation will be investigated with bone Gla protein which will also be a new example of a Ca+2-Gla binding domain.