The VKD proteins VIIa, IXa, Xa and C are are very similar in their catalytic domains--they are all serine proteases with exactly the same general active site. We want to know what subtle differences endow them with specfic reactivity. We hope to do this with the three dimensional models that we have constructed.
Both of these systems are the result of collaborations at NIEHS. The first 3D structures of sulfotransferases came from NIEHS in the late 1990; our involvement has been to take partial results and provide extended models for understanding the function and specificity of these proteins which transfer sulfur (as sulfate) to electronegative atoms on other proteins, carbohydrates or hormones.
The first mammalian P450 x-ray crystal structure was published in 2000 by a Scripps group. This has motivated an effort by us and a group at NIEHS to attempt to organize a significant body of enzymatic data of P450s obtained at NIEHS by constructing 3D models for the specific systems. We are also investigating QM/MM models of the active sites, initially of the heparan sulfotransferases, in collaboration with W. Yang (Duke).
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 prothombinase 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 three aims.
In Aim IV , we will employ still-developing quantum mechanical/molecular mechanical (QM/MM) methodology to the activation of FX by FVIIa/TF and 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 existing experimental 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.