Our laboratory has discovered the Na+-dependent allosteric enhancement of catalytic activity in thrombin and related serine proteases involved in blood coagulation and the complement system, identified the Na+ binding site and elucidated the importance of Na+ binding in the function and evolution of serine proteases. The proposed research project is aimed at gaining a complete functional and structural characterization of thrombin allostery and how it influences recognition of physiological substrates, effectors and inhibitors. We will use a combination of kinetic, mutational and structural studies to identify regions of the enzyme that are under the influence of Na+ binding and constitute the structural scaffold through which the Na+ site communicates changes of functional significance to binding epitopes for physiological ligands. We will determine what regions of the enzyme are involved in recognition of fibrinogen, protein C, the thrombin receptor PAR1, thrombomodulin, heparin and anti- thrombin III with unprecedented detail. The new information to emerged from the proposed studies will broaden our understanding of thrombin interactions in the blood and will enable the identification of molecular targets for new anti-coagulants. We will also exploit the knowledge gained on thrombin allostery to introduce Na+ binding and enhanced catalytic activity in the fibrinolytic enzyme tissue plasminogen activator. These mutational studies will produce more proficient derivatives of the enzyme that may benefit the current treatment of acute myocardial infarction and stroke. Developments from the proposed research plan will impact on the treatment and prevention of thrombotic disorders in which thrombin is directly involved, will have a broad impact on the study of allosteric proteins in general and will demonstrate that proteases with enhanced catalytic activity can be engineered rationally to benefit areas of medical and biotechnological importance.
| Chinnaraj, Mathivanan; Chen, Zhiwei; Pelc, Leslie A et al. (2018) Structure of prothrombin in the closed form reveals new details on the mechanism of activation. Sci Rep 8:2945 |
| Chakraborty, Pradipta; Acquasaliente, Laura; Pelc, Leslie A et al. (2018) Interplay between conformational selection and zymogen activation. Sci Rep 8:4080 |
| Mickevi?i?t?, Aurelija; Timm, David D; Gedgaudas, Marius et al. (2018) Intrinsic thermodynamics of high affinity inhibitor binding to recombinant human carbonic anhydrase IV. Eur Biophys J 47:271-290 |
| Sivaraja, Mohanram; Pozzi, Nicola; Rienzo, Matthew et al. (2018) Reversible covalent direct thrombin inhibitors. PLoS One 13:e0201377 |
| Barranco-Medina, Sergio; Murphy, Mary; Pelc, Leslie et al. (2017) Rational Design of Protein C Activators. Sci Rep 7:44596 |
| Chakraborty, Pradipta; Di Cera, Enrico (2017) Induced Fit Is a Special Case of Conformational Selection. Biochemistry 56:2853-2859 |
| Pozzi, Nicola; Bystranowska, Dominika; Zuo, Xiaobing et al. (2016) Structural Architecture of Prothrombin in Solution Revealed by Single Molecule Spectroscopy. J Biol Chem 291:18107-16 |
| Pozzi, Nicola; Zerbetto, Mirco; Acquasaliente, Laura et al. (2016) Loop Electrostatics Asymmetry Modulates the Preexisting Conformational Equilibrium in Thrombin. Biochemistry 55:3984-94 |
| Eickhoff, Christopher S; Zhang, Xiuli; Vasconcelos, Jose R et al. (2016) Costimulatory Effects of an Immunodominant Parasite Antigen Paradoxically Prevent Induction of Optimal CD8 T Cell Protective Immunity. PLoS Pathog 12:e1005896 |
| Wu, Xiaobin; Kim, Heejeong; Seravalli, Javier et al. (2016) Potassium and the K+/H+ Exchanger Kha1p Promote Binding of Copper to ApoFet3p Multi-copper Ferroxidase. J Biol Chem 291:9796-806 |
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