Investigating the Residue-Level Dynamics of the Thrombin-Thrombomodulin Interaction

Abstract

The serine protease, thrombin catalyzes the final step in the blood clotting cascade by cleaving, and thereby activating, proteins in the blood that consequently form a blood clot. However, thrombin has also demonstrated an anticoagulative role as well, which is initiated by the binding of the cofactor thrombomodulin to thrombin, and is characterized by a change in thrombin substrate affinity away from procoagulative substrates and towards activation of protein C instead. Despite decades of study, the mechanism behind this shift in the substrate preference of thrombin remains unclear. However, evidence from previous studies suggests that this change in activity results from changes in the intermolecular protein dynamics of thrombin that are induced by the binding of thrombomodulin. As a result, the thrombin-thrombomodulin interaction has been suggested to undergo dynamic allostery. Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) experiments have been used to identify the solvent-exposed regions of thrombin that undergo changes in protein dynamics when thrombomodulin is present, yet the limits of the HDX-MS technique prevent the identification of changes at residue-level resolution, as well as any changes in motion of residues buried in the protein core of thrombin. In order to supplement the HDX-MS data collected, Curr-Purcell-Meibloom-Gill (CPMG) NMR experiments have been conducted on S195M mutant thrombin in the presence of thrombomodulin. The results of these experiments will be compared to previous CPMG results published on S195M Mutant thrombin in the absence of thrombomodulin in order to identify which residues are affected by the binding of thrombomodulin to thrombin. Thus, by comparing the rates of transitions to rare states determined from the CPMG experiments, new insights regarding the allosteric transition between thrombin's procoagulative and anticoagulative states will be obtained.