Manna, Rosa
(2016)
Crystallographic studies of coagulation factor XII.
PhD thesis, University of Nottingham.
Abstract
Coagulation Factor XII (FXII) is an important protein involved in the initiation of the intrinsic pathway of the coagulation cascade. Recent studies suggested that FXII may play a role in pathological thrombosis without compromising physiological hemostasis. For this reason, the inhibition of FXII could represent a new and selective strategy for preventing stroke and other thromboembolic diseases. Since there is no structure available for the protease domain of FXII, the knowledge of the overall domain structure can be significantly helpful in the development of inhibitors with specific selectivity for the target.
Three different constructs were made: βFXIIa, βFXIIG570R, a missense mutations that causes a reduced activity of FXIIa, and βFXIIa fused with Maltose Binding Protein (MBP) at the N-terminus. An expression and purification protocol was established for all the proteins. In order to assess the activity of the recombinant protein, the hydrolysis of chromogenic substrate S2302 was measured and a comparison with commercial αFXIIa and βFXIIa was performed. The kinetic parameters (kcat, KM, kcat/KM, Vmax) indicated that the catalytic behaviour of recombinant MBP-βFXIIa and βFXIIa is essentially identical to that of commercial βFXIIa purified from plasma. Therefore, the protein expressed and purified from insect cell system is catalytically competent.
Large efforts were devoted to the identification of possible crystallization conditions for the expressed and purified constructs, in complex with different inhibitors, both small molecules and macromolecular inhibitors. Diffracting crystals were obtained for MBP-βFXIIa in complex with D-Phe-Pro-Argchloromethylketone (PPACK). A complete data set was collected at 4 Å. Notwithstanding the low resolution of the data, the structural analysis of key elements and the comparison with the zymogen confirmed that recombinant protein is in the active conformation. Moreover, a first analysis of the structure and of the lattice packing enabled to understand some key differences between the interactions that PPACK forms when in complex to FXIIa and with thrombin, thus possible explaining the lower affinity of PPACK for FXIIa. This is the first crystallographic structure of FXIIa and it represents a first step in the study of protein-inhibitor interactions from structural consideration.
βFXII construct was also cloned in a different vector for the expression in E. coli. The cleavage of TF tag was performed using three different enzymes (HRV-3C protease, FXa and thrombin). The best results were obtained, using the thrombin and setting the reaction overnight at room temperature. However, it was not possible to continue the process, since the protein was lost after the cleavage. This could be due to a poor stability of the protein. For that reason, a different strategy was developed, consisting of co-purification of βFXII with Ecotin. The presence of the inhibitor was thought to stabilize the protein during the cleavage. However, the gel filtration revealed that the complex was not formed.
The possibility to investigate at atomic level the interactions of the inhibitors with FXII could allow an understanding as to how the substrate-inhibitor recognition mechanism work. This could therefore represent a good starting point for the development of new inhibitors that have higher specificity for FXII.
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