Ceresa E, De Maeyer M, Jonckheer A, Peeters M, Engelborghs Y, Declerck P J, Gils A
Laboratory for Pharmaceutical Biology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium.
J Thromb Haemost. 2007 Oct;5(10):2105-12. doi: 10.1111/j.1538-7836.2007.02720.x. Epub 2007 Jul 31.
Activated thrombin activatable fibrinolysis inhibitor (TAFIa) plays a pivotal role in fibrinolysis. TAFIa activity is regulated by a temperature-dependent instability. This instability has not only complicated the study of structure-function relationships of TAFIa but has also prevented the crystallization of TAFIa. Furthermore, the TAFIa instability has severely compromised the development of activity inhibiting monoclonal antibodies. Recently, we combined all known stabilizing mutations (i.e. S305C, T325I, T329I, H333Y and H335Q) resulting in a synergistic (one hundred and eightyfold) stabilization of TAFIa at 37 degrees C. All these residues are located in an amino acid region (AA297-335) consisting of alpha-helix 9 and beta-sheet 11.
To provide a comparative evaluation of the characteristics of a panel of stable TAFIa mutants and an energy-minimized model of the most stable TAFI variant.
The catalytic efficiency for activation of TAFI by thrombin/thrombomodulin was higher for all TAFI mutants compared with TAFI-wild type (wt). Except for TAFI variants carrying T325I-T329I, S305C-T325I or S305C-T325I-T329I mutations, the catalytic efficiency for Hip-Arg hydrolysis by TAFIa was similar for the TAFI mutants compared with the wild type. All TAFIa variants were equally well inhibited by potato tuber carboxypeptidase inhibitor (PTCI) and showed a significantly increased antifibrinolytic potential in accordance with their increased stability. Based on the intrinsic fluorescence decay of TAFIa, two independent structural transitions were found to be associated with the loss of functional activity.
Using molecular dynamic calculations on both TAFI-wt and TAFI-S305C-T325I-T329I-H333Y-H335Q models, we were able to identify the molecular interactions that contribute to the increased stability of the mutants.
活化的凝血酶激活的纤维蛋白溶解抑制剂（TAFIa）在纤维蛋白溶解过程中起关键作用。TAFIa的活性受温度依赖性不稳定性的调节。这种不稳定性不仅使TAFIa的结构-功能关系研究变得复杂，还阻碍了TAFIa的结晶。此外，TAFIa的不稳定性严重影响了抑制其活性的单克隆抗体的研发。最近，我们将所有已知的稳定突变（即S305C、T325I、T329I、H333Y和H335Q）组合在一起，使TAFIa在37℃时实现了协同（180倍）稳定。所有这些残基都位于由α-螺旋9和β-折叠11组成的氨基酸区域（AA297-335）中。
对一组稳定的TAFIa突变体的特性以及最稳定的TAFI变体的能量最小化模型进行比较评估。
与野生型TAFI（wt）相比，所有TAFI突变体被凝血酶/血栓调节蛋白激活TAFI的催化效率更高。除了携带T325I-T329I、S305C-T325I或S305C-T325I-T329I突变的TAFI变体，与野生型相比，TAFI突变体的TAFIa水解Hip-Arg的催化效率相似。所有TAFIa变体均被马铃薯块茎羧肽酶抑制剂（PTCI）同等程度地抑制，并且根据其增加的稳定性显示出显著增加的抗纤维蛋白溶解潜力。基于TAFIa的固有荧光衰减，发现两个独立的结构转变与功能活性的丧失相关。
通过对TAFI-wt和TAFI-S305C-T325I-T329I-H333Y-H335Q模型进行分子动力学计算，我们能够确定有助于突变体稳定性增加的分子相互作用。
