Department of Biological Sciences, 16 Science Drive 4, National University of Singapore, Singapore 117558.
Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117600.
Biosci Rep. 2022 May 27;42(5). doi: 10.1042/BSR20220421.
Kazal-type protease inhibitors strictly regulate Factor XIIa (FXIIa), a blood-clotting serine protease. However, when negatively charged surface of prosthetic device come into contact with FXII, it undergoes conformational change and auto-activation, leading to thrombus formation. Some research suggests that Kazal-type protease inhibitor specificity against FXIIa is governed solely by the reactive-site loop sequence, as this sequence makes most-if not all-of the direct contacts with FXIIa. Here, we sought to compare the inhibitory properties of two Kazal-type inhibitors, Infestin-4 (Inf4), a potent inhibitor of FXIIa, and Aedes aegypti trypsin inhibitor (AaTI), which does not inhibit FXIIa, to better understand Kazal-type protease specificity and determine the structural components responsible for inhibition. There are only three residue differences in the reactive-site loop between AaTI and Inf4. Through site-directed mutagenesis, we show that the reactive-site loop is only partially responsible for the inhibitory specificity of these proteases. The protein scaffold of AaTI is unstable due to an elongated C5C6 region. Through chimeric study, we show that swapping the protease-binding loop and the C5C6 region from Inf4 with that of AaTI can partially enhance the inhibitory activity of the AaTI_Inf4 chimera. Furthermore, the additional substitution of Asn at the P14' position of AaTI with Gly (Gly27 in Inf4) absolves the steric clashing between AaTI and the surface 140-loop of FXIIa, and increases the inhibition of the chimeric AaTI to match that of wild-type Inf4. Our findings suggest that ancillary regions in addition to the reactive-site loop sequence are important factors driving Kazal-type inhibitor specificity.
Kazal 型蛋白酶抑制剂严格调节凝血酶 FXIIa，然而，当假体表面与 FXII 接触时，它会发生构象变化和自动激活，导致血栓形成。一些研究表明，Kazal 型蛋白酶抑制剂对 FXIIa 的特异性仅受反应位点环序列控制，因为该序列与 FXIIa 直接接触最多（如果不是全部）。在这里，我们试图比较两种 Kazal 型抑制剂的抑制特性，Infestin-4（Inf4），一种有效的 FXIIa 抑制剂，和埃及伊蚊胰蛋白酶抑制剂（AaTI），它不抑制 FXIIa，以更好地了解 Kazal 型蛋白酶的特异性，并确定负责抑制的结构成分。在 AaTI 和 Inf4 的反应位点环之间只有三个残基差异。通过定点突变，我们表明反应位点环仅部分负责这些蛋白酶的抑制特异性。由于 C5C6 区域的伸长，AaTI 的蛋白质支架不稳定。通过嵌合研究，我们表明从 Inf4 交换蛋白酶结合环和 C5C6 区域与 AaTI 的可以部分增强 AaTI_Inf4 嵌合体的抑制活性。此外，在 AaTI 的 P14'位置用 Gly（Inf4 中的 Gly27）取代额外的 Asn，消除了 AaTI 与 FXIIa 表面 140 环之间的空间冲突，并增加了嵌合 AaTI 的抑制作用，使其与野生型 Inf4 相匹配。我们的研究结果表明，除了反应位点环序列之外，辅助区域也是驱动 Kazal 型抑制剂特异性的重要因素。
