Abstract
Trypsin-like proteases are synthesized as zymogens and activated through a mechanism that folds the active site for efficient binding and catalysis. Ligand binding to the active site is therefore a valuable source of information on the changes that accompany zymogen activation. Using the physiologically relevant transition of the clotting zymogen prothrombin to the mature protease thrombin, we show that the mechanism of ligand recognition follows selection within a pre-existing ensemble of conformations with the active site accessible (E) or inaccessible (E*) to binding. Prothrombin exists mainly in the E* conformational ensemble and conversion to thrombin produces two dominant changes: a progressive shift toward the E conformational ensemble triggered by removal of the auxiliary domains upon cleavage at R271 and a drastic drop of the rate of ligand dissociation from the active site triggered by cleavage at R320. Together, these effects produce a significant (700-fold) increase in binding affinity. Limited proteolysis reveals how the E*-E equilibrium shifts during prothrombin activation and influences exposure of the sites of cleavage at R271 and R320. These new findings on the molecular underpinnings of prothrombin activation are relevant to other zymogens with modular assembly involved in blood coagulation, complement and fibrinolysis.