Thrombomodulin (TM) is an essential anticoagulant protein that binds to thrombin and the resulting complex shuts down further production of thrombin. In the complex, the thrombin active site has new substrate specificity toward protein C, which when activated by the thrombin- TM complex shuts down the coagulation cascade. The proposed experiments will analyze both the structure and function of TM in order to obtain an atomic-level understanding of how TM alters the activity of thrombin. Structural information gained from multi-dimensional NMR methods as well as X-ray crystallography will be integrated with functional information gained from site-directed mutagenesis of residues in both TM and thrombin. Particular emphasis will be placed on the nature of the protein-protein interaction and on the conformational changes that occur during the interaction. The experimental plan is as follows: 1) Determine the structure of the smallest active fragment of TM , TMEGF(4-5) using multi-dimensional NMR methods. 2) Determine the function of essential residues within the fourth EGF-like domain of TM. Essential residues that have been shown to coalesce in a clefT on the fourth domain will be conservatively mutated and analyzed for functional changes using clotting, protein C activation, and direct binding assays. 3) Determine the function of essential residues within the fifth EGF-like domain of TM. Essential residues within the C-terminal loop and tail will be conservatively mutated and each mutant will be analyzed for functional changes as in Aim 2. 4) Determine the structural consequences of interesting functionally altered mutants using methods developed in Aim 1. 5) Express the S195A mutant of human prothrombin-2 in large quantities in Pichia pastoris. This mutant thrombin is required for both NMR and X-ray crystallographic studies. 6) Determine the surface of TMEGF(4-5) that interacts with thrombin using a combination of X-ray crystallography and isotope-edited NMR as well as complementary mutagenesis of residues on both TM and thrombin.
| Mentz, S; de Lacalle, S; Baerga-Ortiz, A et al. (1999) Mechanism of thrombin clearance by human astrocytoma cells. J Neurochem 72:980-7 |
