X-RAY CRYSTALLOGRAPHY

Alexander Tulinsky

University Distinguished Professor Emeritus (b. 1928). B.A., 1952, Temple University; Ph.D., 1956, Princeton University; Research Associate, 1955-59, Protein Structure Project. Physical Chemistry and X-ray Crystallography. Structure of proteins and enzymes of blood coagulation and fibrinolysis.

517-355-9715, Ext. 250

tulinsky@cem.msu.edu

 

Our research program centers around the structure determination of molecules of blood coagulation and fibrinolysis (prothrombin and some of its larger activation products, fragments of plasminogen, thrombin, antithrombin, factor X, etc.). We also use the X-ray crystallographic method to carry out chemical and biochemical investigations that characterize the expression of biological functionality through derivative structure determinations: 1) chemically modified and irreversibly inhibited enzyme, 2) competitive, reversible, equilibrium inhibition 3) inhibition with substrate and transition state analogues and catalysis products, 4) ligand binding.

The general thrust of the long range plans is to pursue crystallographically, structural aspects of molecules of blood coagulation and fibrinolysis with the aim of relating them to functionality at the molecular level. Our research program has been exclusively devoted to the area for about the past 20 years and has made significant progress by determining the structures of all but one of the autonomous domains of these molecules (g-carboxyglutamic, kringle, epidermal growth factor, catalytic) along with many informative derivative studies involving them or combinations thereof. We are now extending the work to include additional catalytically active multidomain structures, which will also address domain-domain associations in a more general way. Since these molecules interact with macromolecular substrates, inhibitors and cofactors, the manner of such interactions will be revealed through the structure determinations of molecular complexes, including rationally designed mimetic molecules. Specific studies targeted include: 1) the inactive precursor of a-thrombin, 2) Ser195Ala anhydro thrombin and hirugen-anhydro thrombin to capture the active site conformational change accompanying fibrinogen recognition exosite binding, 3) a conformationally restricted non-peptide mimetic based on the unusual N-terminal hirudin interaction in the active site of thrombin, 4) the structure determination of Factor Xa in the presence of Ca+2 ions and in complex with the second Kunitz domain of tissue factor pathway inhibitor, 5) the structure determination of the potent Factor Xa inhibitor tick anticoagulant protein, 6) kringle-kringle interactions, 7) epidermal growth factor-like (EGF)-kringle interactions and, 8) EGF-EGF and EGF-catalytic domain associations. Macromolecular interactions with carbohydrates will be determined through low molecular weight heparin complexes with thrombin and the kringle of urokinase. Crystallization searches are also underway with Factors VIIa and IX and protein C and activated protein C to extend the scope of the work to other areas of the blood coagulation cascade. Diffraction quality single crystals of many of the above have been grown and their X-ray diffraction patterns examined in a preliminary way.

Representative Publications

The Molecular Environment of the Na+ Binding Site of Thrombin, E. Zhang and A. Tulinsky, Biophys. Chem., 63, 185 (1997).

Crystal Structures of Thrombin and Thiazole-Containing Inhibitors as Probes of the S1' Binding Site, J. H. Matthews, R. Krishnan, M. J. Constanzo, B. Maryanoff, and A. Tulinsky, Biophys. J., 71, 2830 (1996).

Molecular Interactions of Thrombin, A. Tulinsky, Sem. Thromb.,Hemost., 22, 117-123 (1996).

Comparison of the Structures of the Cycletheonamide A Complexes of Human a-Thrombin and Bovine b-Trypsin, V. Ganesh, A. Y. Lee, J. Clardy, and A. Tulinsky, Prot. Sci., 5, 825 (1996).

The Crystal Structures of the Recombinant Kringle1 Domain of Human Plasminogen in Complexes with the Ligands, Aminocaproic Acid and trans-4-aminomethylcyclohexane-1-Carboxylic Acid, I. I. Mathews, P. Vanderhoff-Hanaver, F. J. Castellino, and A. Tulinsky, Biochemistry, 35, 2567 (1996).