Structure-function study of blood coagulation factor VII by in vitro mutagenesis and computer simulation.
详细信息
文摘
Coagulation factor VIIa (FVIIa) is a plasma trypsin-like protease. It consists of a γ-carboxyl glutamic acid domain (Gla), 2 epidermal growth factor-like domains (Egf1 and Egf2) and a catalytic domain at its C-terminus. Activation of FVII to FVIIa, followed by binding to its cofactor, tissue factor (TF), is the first step in the extrinsic pathway of the blood coagulation cascade. I investigated the following questions: what FVII residues are important for activation, what FVIIa residues are involved in macromolecular substrate binding and what is the mechanism by which TF enhances FVIIa's activity? To address these questions, I used molecular simulation and FVIIa 3-D structure information to identify potentially important residues and employed molecular biological methods to create mutant FVII's. I found a hydrophobic patch on the surface of FVII's catalytic domain which is specifically involved in the activation of FVII by factor Xa (FXa). Mutation of two residues in this patch, H101 and Y179 to alanine (H101A, Y179A) decreased the catalytic efficiencies of FXa in FVII activation by 15- and 8-fold respectively. Activation of FVII with multiple mutations including both H101A and Y179A by FXa was not measurable. Furthermore, I identified an exosite involved in FX and FIX binding on the surface of FVIIa, about 90° from the hydrophobic patch. Mutations at this exosite, L144Aa and R147Aa, had kcat's lower than wild-type by 40- and 4-fold respectively toward FX Activation of FIX was also affected by these mutations, although less significantly than FX activation. A FVIIa with mutations including both L144A and R147 had almost no activity for FX or FIX activation, indicating that FX and FIX share at least part of the same binding site on FVIIa's surface. Because the Egf1 domain contributes to TF binding, I used FVII with its Egf1 domain substituted with FIX's (FVII IXegf1) to study Egf1's function. in addition to TF binding, the catalytic activity of FVIIa was affected. Mutation of the residue (lysine) at position 79 back to FVII's (arginine) regained some functions both in TF binding affinity and its catalytic activity. The affected catalytic activities were displayed only in the presence of TF. My hypothesis is that the Egf1 substitution effect is transmitted through the interface between the catalytic domain and the N-terminus of TF. To test this hypothesis, I carried out dynamics simulation of FVIIa-TF and FVIIIXegf1a-TF complexes. The results showed that 3 loops, with residues 310–329, residues 333–337 and residues 369–374, were deformed and shifted in position due to the disruption of the catalytic domain-TF interface. This affected both FVII's substrate binding sites and the hydrophobic environment required for stabilizing the N-terminus insertion of the catalytic domain of FVIIa. The simulations suggest that any changes in the local conformation of these loops or their neighborhood may affect catalytic activity. This prediction is supported by the evidence from in vitro mutagenesis.