Function and Dynamics of Thrombin by NMR

Abstract

Thrombin is a serine protease that plays an essential role in the blood-clotting cascade, cleaving soluble fibrinogen to insoluble fibrin and activating platelets. Thrombin is also involved in tissue repair, angiogenesis and inflammation via the activation of the PAR-1 and PAR-3 receptors. In its positive feedback loop, thrombin activates factors XI, V, and VIII to promote further clotting. When thrombin binds thrombomodulin (TM) at anion binding exosite 1 (ABE1) the feedback loop becomes negative by activating protein C, which in turn inactivates factors VIIIa and Va, decreasing the rate of thrombin activation. The mechanism of the thrombin specificity change from procoagulant to anticoagulant remains elusive since crystal structures of thrombin bound to TM show no structural change in the active site. NMR provides the residue specific resolution needed to study the dynamics and the contribution to thrombin function. Near complete assignments of thrombin backbone resonances have been achieved using high-field TROSY based methods to study [2H,15N,13C]thrombin. Relaxation and relaxation dispersion experiments performed on thrombin covalently inhibited with PPACK show functional regions with increased dynamics, including ABE1, the γ-loop, and ABE 2. Analysis of residual dipolar couplings in light of accelerated molecular dynamics simulations indicated a structural ensemble that is highly mobile in several timescales, providing a path forward towards understanding the role of dynamics and conformational fluctuations on catalysis and substrate interactions in thrombin.