Abstract
Prothrombin, or coagulation factor II, is a multidomain zymogen precursor of thrombin that undergoes an allosteric equilibrium between two alternative conformations, open and closed, that react differently with the physiological activator prothrombinase. Specifically, the dominant closed form promotes cleavage at R320 and initiates activation along the meizothrombin pathway, whilst the open form promotes cleavage at R271 and initiates activation along the alternative prethrombin-2 pathway. Here we report how key structural features of prothrombin can be monitored by limited proteolysis with chymotrypsin that attacks W468 in the flexible autolysis loop of the protease domain in the open but not the closed form. Perturbation of prothrombin by selective removal of its constituent Gla domain, kringles and linkers reveals their long-range communication and supports a scenario where stabilization of the open form switches the pathway of activation from meizothrombin to prethrombin-2. We also identify R296 in the A chain of the protease domain as a critical link between the allosteric open-closed equilibrium and exposure of the sites of cleavage at R271 and R320. These findings reveal important new details on the molecular basis of prothrombin function.