Structural And Functional Studies On Thrombin Allostery

Thrombin is an allosteric serine protease endowed with important physiological functions in the blood. Once activated, α-thrombin plays procoagulant, prothrombotic, and anticoagulant functions in blood. Its important role in blood coagulation can be emphasized by the fact that thrombin interacts with numerous substrates, cofactors, ligands, and inhibitors. Two well-documented allosteric pathways, involving the Na+ binding site and the exosite I, regulate thrombin catalytic efficiency and substrate specificity. Identifying structural epitopes of substrate binding, the exact pathway and the molecular basis of allosteric regulation remains challenging. The case of protease-activated receptor: PAR1) is particularly relevant in view of the plethora of biological effects associated with its activation by thrombin. We present the crystal structure of thrombin in complex with a 30-residue long uncleaved extracellular fragment of PAR1. The structure reveals novel and unexpected features of thrombin-PAR1 interaction, paving the way for future protein engineering studies. Furthermore, thrombin mutants D102N and WE are stabilized in a self-inhibited conformation, E*, where access to the active site is occluded by a collapse of the entire 215-219 β-strand. Binding of PAR1 fragment to exosite I, 30-Ã… away from the active site region, causes a large conformational change and restores access to the active site, akin to the E form in the D102N mutant but not the WE mutant. Using a network of polar interactions, we propose the structural basis of a likely long-range allosteric communication in thrombin. These structural findings, along with kinetic and calorimetry data, indicate that WE is strongly stabilized in the E* form and reveals the molecular basis of its potent anticoagulant property in vivo. We have developed a new expression system using E. coli to label thrombin for solution based structural studies. 19F-NMR results indicate that α-thrombin is a highly dynamic and flexible molecule. Binding of Na+, an exosite I ligand, and an irreversible inhibitor induces significant and unique structural and dynamic changes. A zymogen form of thrombin, prethrombin-2, displays unique structural signatures when compared to α-thrombin. 19F-NMR results corroborate our recent crystallographic data, expand the repertoire obtained from previous x-ray structural studies and highlight the unprecedented and previously undocumented plasticity endowed to a thrombin molecule. Taken together, results presented in this thesis fill a significant gap in our understanding of the molecular mechanisms of substrate recognition by thrombin and its allosteric regulation.

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biology and Biomedical Sciences: Computational and Molecular Biophysics

Year dc:date.available

2010

Author dc:creator

• Gandhi, Prafull

Contributors dc:contributor

• Enrico Di Cera

• Biochemistry
• Biophysics
• Allostery
• Isothermal titration calorimetry
• Protease activated receptors
• protein structure and function
• Thrombin
• X-ray crystallography

Language dc:language

English (en)

DOI

https://doi.org/10.7936/K79021SH

OAI identifier oai:identifier

oai:openscholarship.wustl.edu:etd-1847