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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL025942-07
Application #
3338369
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1988-12-01
Project End
1993-11-30
Budget Start
1988-12-01
Budget End
1989-11-30
Support Year
7
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Michigan State University
Department
Type
Schools of Arts and Sciences
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
Rios-Steiner, Jorge L; Murakami, Mario T; Tulinsky, Alexander et al. (2007) Active and exo-site inhibition of human factor Xa: structure of des-Gla factor Xa inhibited by NAP5, a potent nematode anticoagulant protein from Ancylostoma caninum. J Mol Biol 371:774-86
Abad, Marta C; Arni, R K; Grella, Davida K et al. (2002) The X-ray crystallographic structure of the angiogenesis inhibitor angiostatin. J Mol Biol 318:1009-17
Rios-Steiner, J L; Schenone, M; Mochalkin, I et al. (2001) Structure and binding determinants of the recombinant kringle-2 domain of human plasminogen to an internal peptide from a group A Streptococcal surface protein. J Mol Biol 308:705-19
St Charles, R; Padmanabhan, K; Arni, R V et al. (2000) Structure of tick anticoagulant peptide at 1.6 A resolution complexed with bovine pancreatic trypsin inhibitor. Protein Sci 9:265-72
Arni, R K; Padmanabhan, K P; Tulinsky, A (1999) Crystallization and preliminary diffraction data of a platelet-aggregation inhibitor from the venom of Agkistrodon piscivorus piscivorus (North American water moccasin). Acta Crystallogr D Biol Crystallogr 55:1468-70
Zhang, E; St Charles, R; Tulinsky, A (1999) Structure of extracellular tissue factor complexed with factor VIIa inhibited with a BPTI mutant. J Mol Biol 285:2089-104
Mochalkin, I; Cheng, B; Klezovitch, O et al. (1999) Recombinant kringle IV-10 modules of human apolipoprotein(a): structure, ligand binding modes, and biological relevance. Biochemistry 38:1990-8
Chang, Y; Mochalkin, I; McCance, S G et al. (1998) Structure and ligand binding determinants of the recombinant kringle 5 domain of human plasminogen. Biochemistry 37:3258-71
Sabharwal, A K; Padmanabhan, K; Tulinsky, A et al. (1997) Interaction of calcium with native and decarboxylated human factor X. Effect of proteolysis in the autolysis loop on catalytic efficiency and factor Va binding. J Biol Chem 272:22037-45
Ganesh, V; Lee, A Y; Clardy, J et al. (1996) Comparison of the structures of the cyclotheonamide A complexes of human alpha-thrombin and bovine beta-trypsin. Protein Sci 5:825-35

Showing the most recent 10 out of 37 publications