We propose to leverage recent advances in computer technology and algorithm development, together with the growing database of three-dimensional (3D) structures of Vitamin K-dependent (VKD) proteins and their complexes, to build reliable 3D complexes in electrically neutral solvent and with structural water molecules in place. It is our hypothesis that theoretical techniques are now at a level of sophistication and accuracy to warrant the careful application to key coagulation systems: the extrinsic tenase complex, which initiates the extrinsic blood coagulation cascade, the prothrombinase complex, which leads to the penultimate step in the cascade (formation of thrombin) and the Factor Xa inhibition complex, which affects the pool of available Factor Xa for thrombin formation. We propose to provide solvent-equilibrated models for these systems via AIM I: Factor VIIa/Tissue Factor (FVIIa/TF) with Factors X and IX and their activated forms and of FVIIa/TF/FX with Tissue Factor Pathway Inhibitor (TFPI);
AIM II : prothrombin(II); II/FXa , and II/FXa/Factor Va;
AIM III : Protein Z (PZ), its complex with FXa and PZ/FXa/Protein Z inhibitor (ZPI). The influence of a membrane surface on complex organization will be considered for each of these aims.
In Aim I V, we will employ still-developing quantum mechanical/molecular mechanical (QM/MM) methodology to the activation of FX by FVIIa/TFand to the transfer of sulfate from PAPS, the ubiquitous source of sulfate in biological systems, by heparan sulfotransferase. These two systems are ideal because of the quality of the existingexperimental structural data and the value that can be derived from understanding the details of the reactions of these particular systems.
The final aim recognizes the need to not only develop all atom, solvated 3D structures, but also to provide insight into the quantum mechanical bond-breaking and bond-forming mechanisms that regulate coagulation. The developed complex structures will be made available through the internet and these will maintain value as a base for systematic improvement even as new experimental structures are solved.
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