Structural enzymology of factor V activation

Abstract Work under previous support has broadened our understanding of the factors that influence the catalytic activity of thrombin and set the stage for a structure-based characterization of how this enzyme interacts with physiological substrates. Unraveling the architecture of factors involved in blood coagulation remains a challenging task because of the difficulty of obtaining high resolution structures for proteins containing multiple domains. We have recently shown how to address this challenge by using cryo-EM, the new gold standard for the structural investigation of biological macromolecules. The proposed research project is a segue to our pioneering cryo-EM structural work on human coagulation factors V and Va free and bound to factor Xa in the prothrombinase complex and the structure of this complex bound to prothrombin. Specifically, we plan to solve the cryo-EM structures of factor V in complex with thrombin (aim 1) and its active precursor meizothrombin (aim 2) with the goal of revealing the molecular basis of a key step of the initiation phase of the coagulation response that leads to assembly of the prothrombinase complex. The proposal is supported by exciting preliminary cryo- EM maps currently refined at 6.6 Å resolution for the thrombin-factor V complex (aim 1) and 3.8 Å resolution for the meizothrombin-fV complex (aim 2). Once fully refined, these structures will reveal the distinct modes of binding of thrombin and meizothrombin at preferred sites of activation and the full architecture of meizothrombin for the first time. Underlying epitopes will be validated independently by mutagenesis of specific residues of thrombin, meizothrombin and factor V. In addition, the structures will test the hypothesis that binding of thrombin and meizothrombin to factor V rigidifies the disordered B domain and visualizes the structural determinants that keep factor V in its inactive state. Success of the proposed studies will significantly advance our basic knowledge on factor V and its activation in ways that are directly relevant to other multidomain proteins in the blood coagulation cascade and that may benefit the development of new therapeutic strategies.