
Enrico Di Cera, M.D.
Alice A. Doisy Professor and Chairman
Interim Vice Dean for Research
Structural enzymology of coagulation factors.
Research Interests
We are interested in the interaction of prothrombin with prothrombinase leading to generation of thrombin and blood clotting, and the interaction of the thrombin-thrombomodulin complex with protein C initiating the negative feed-back loop that shuts down the coagulation response. Our experimental approach merges classical enzymology and X-ray structural biology with innovative techniques like smFRET, 19F NMR and cryo-EM.
Research Highlights
Selected Reviews:
1. Site-specific analysis of mutational effects in proteins. Di Cera E. (1998) Adv Protein Chem 51, 59-119.
2. Evolution of enzyme cascades from embryonic development to blood coagulation. Krem MM, Di Cera E. (2002) Trends Biochem Sci 26, 67-74.
Recent Publications
Mechanisms of ligand binding
Mechanisms of ligand binding
Many processes in chemistry and biology involve interactions of a ligand with its molecular target. Interest in the mechanism governing such interactions has dominated theoretical and experimental analysis for over a century. The interpretation of molecular recognition has evolved from a simple rigid body association of the ligand with its target to appreciation of the key role played by conformational transitions. Two conceptually distinct descriptions have had a profound impact on our understanding of mechanisms of ligand binding. The first description, referred to as induced fit, assumes that conformational changes follow the initial binding step to optimize the complex between the ligand and its target. The second description, referred to as conformational selection, assumes that the free target exists in multiple conformations in equilibrium and that the ligand selects the optimal one for binding. Both descriptions can be merged into more complex reaction schemes that better describe the functional repertoire of macromolecular systems. This review deals with basic mechanisms of ligand binding, with special emphasis on induced fit, conformational selection, and their mathematical foundations to provide rigorous context for the analysis and interpretation of experimental data. We show that conformational selection is a surprisingly versatile mechanism that includes induced fit as a mathematical special case and even captures kinetic properties of more complex reaction schemes. These features make conformational selection a dominant mechanism of molecular recognition in biology, consistent with the rich conformational landscape accessible to biological macromolecules being unraveled by structural biology.
Zymogen and activated protein C have similar structural architecture
Zymogen and activated protein C have similar structural architecture
Activated protein C is a trypsin-like protease with anticoagulant and cytoprotective properties that is generated by thrombin from the zymogen precursor protein C in a reaction greatly accelerated by the cofactor thrombomodulin. The molecular details of this activation remain elusive due to the lack of structural information. We now fill this gap by providing information on the overall structural organization of these proteins using single molecule FRET and small angle X-ray scattering. Under physiological conditions, both zymogen and protease adopt a conformation with all domains vertically aligned along an axis 76 Å long and maximal particle size of 120 Å. This conformation is stabilized by binding of Ca to the Gla domain and is affected minimally by interaction with thrombin. Hence, the zymogen protein C likely interacts with the thrombin-thrombomodulin complex through a rigid body association that produces a protease with essentially the same structural architecture. This scenario stands in contrast to an analogous reaction in the coagulation cascade where conversion of the zymogen prothrombin to the protease meizothrombin by the prothrombinase complex is linked to a large conformational transition of the entire protein. The presence of rigid epidermal growth factor domains in protein C as opposed to kringles in prothrombin likely accounts for the different conformational plasticity of the two zymogens. The new structural features reported here for protein C have general relevance to vitamin K-dependent clotting factors containing epidermal growth factor domains, such as factors VII, IX, and X.
Role of the activation peptide in the mechanism of protein C activation
Role of the activation peptide in the mechanism of protein C activation
Protein C is a natural anticoagulant activated by thrombin in a reaction accelerated by the cofactor thrombomodulin. The zymogen to protease conversion of protein C involves removal of a short activation peptide that, relative to the analogous sequence present in other vitamin K-dependent proteins, contains a disproportionately high number of acidic residues. Through a combination of bioinformatic, mutagenesis and kinetic approaches we demonstrate that the peculiar clustering of acidic residues increases the intrinsic disorder propensity of the activation peptide and adversely affects the rate of activation. Charge neutralization of the acidic residues in the activation peptide through Ala mutagenesis results in a mutant activated by thrombin significantly faster than wild type. Importantly, the mutant is also activated effectively by other coagulation factors, suggesting that the acidic cluster serves a protective role against unwanted proteolysis by endogenous proteases. We have also identified an important H-bond between residues T176 and Y226 that is critical to transduce the inhibitory effect of Ca and the stimulatory effect of thrombomodulin on the rate of zymogen activation. These findings offer new insights on the role of the activation peptide in the function of protein C.
VE-1902-A direct thrombin inhibitor with reversible covalent mechanism of action shows efficacy with reduced bleeding in rodent models of thrombosis
VE-1902-A direct thrombin inhibitor with reversible covalent mechanism of action shows efficacy with reduced bleeding in rodent models of thrombosis
High incidence of bleeding events remains a key risk for patients taking anticoagulants, especially those in need of long-term combination therapy with antiplatelet agents. As a consequence, patients may not receive clinically indicated combination antithrombotic therapy. Here, we report on VE-1902, a member of a novel class of precision oral anticoagulants (PROACs) that combines effective anticoagulation with reduced bleeding in preclinical testing.
F NMR reveals the conformational properties of free thrombin and its zymogen precursor prethrombin-2
F NMR reveals the conformational properties of free thrombin and its zymogen precursor prethrombin-2
The conformational properties of trypsin-like proteases and their zymogen forms remain controversial because of a lack of sufficient information on their free forms. Specifically, it is unclear whether the free protease is zymogen-like and shifts to its mature form upon a ligand-induced fit or exists in multiple conformations in equilibrium from which the ligand selects the optimal fit via conformational selection. Here we report the results of F NMR measurements that reveal the conformational properties of a protease and its zymogen precursor in the free form. Using the trypsin-like, clotting protease thrombin as a relevant model system, we show that its conformation is quite different from that of its direct zymogen precursor prethrombin-2 and more similar to that of its fully active Na-bound form. The results cast doubts on recent hypotheses that free thrombin is zymogen-like and transitions to protease-like forms upon ligand binding. Rather, they validate the scenario emerged from previous findings of X-ray crystallography and rapid kinetics supporting a pre-existing equilibrium between open (E) and closed (E*) forms of the active site. In this scenario, prethrombin-2 is more dynamic and exists predominantly in the E* form, whereas thrombin is more rigid and exists predominantly in the E form. Ligand binding to thrombin takes place exclusively in the E form without significant changes in the overall conformation. In summary, these results disclose the structural architecture of the free forms of thrombin and prethrombin-2, consistent with an E*-E equilibrium and providing no evidence that free thrombin is zymogen-like.