Fodor Krisztián, Harmat Veronika, Hetényi Csaba, Kardos József, Antal József, Perczel András, Patthy András, Katona Gergely, Gráf László
Biotechnology Research Group of the Hungarian Academy of Sciences, Eötvös Loránd University, Budapest, H-1117, Hungary.
J Mol Biol. 2005 Jul 1;350(1):156-69. doi: 10.1016/j.jmb.2005.04.039.
We have previously shown that a trypsin inhibitor from desert locust Schistocerca gregaria (SGTI) is a taxon-specific inhibitor that inhibits arthropod trypsins, such as crayfish trypsin, five orders of magnitude more effectively than mammalian trypsins. Thermal denaturation experiments, presented here, confirm the inhibition kinetics studies; upon addition of SGTI the melting temperatures of crayfish and bovine trypsins increased 27 degrees C and 4.5 degrees C, respectively. To explore the structural features responsible for this taxon specificity we crystallized natural crayfish trypsin in complex with chemically synthesized SGTI. This is the first X-ray structure of an arthropod trypsin and also the highest resolution (1.2A) structure of a trypsin-protein inhibitor complex reported so far. Structural data show that in addition to the primary binding loop, residues P3-P3' of SGTI, the interactions between SGTI and the crayfish enzyme are also extended over the P12-P4 and P4'-P5' regions. This is partly due to a structural change of region P10-P4 in the SGTI structure induced by binding of the inhibitor to crayfish trypsin. The comparison of SGTI-crayfish trypsin and SGTI-bovine trypsin complexes by structure-based calculations revealed a significant interaction energy surplus for the SGTI-crayfish trypsin complex distributed over the entire binding region. The new regions that account for stronger and more specific binding of SGTI to crayfish than to bovine trypsin offer new inhibitor sites to engineer in order to develop efficient and specific protease inhibitors for practical use.
我们之前已经表明，沙漠蝗虫（Schistocerca gregaria）的一种胰蛋白酶抑制剂（SGTI）是一种分类群特异性抑制剂，它抑制节肢动物胰蛋白酶，如小龙虾胰蛋白酶的效果比哺乳动物胰蛋白酶高五个数量级。本文进行的热变性实验证实了抑制动力学研究结果；加入SGTI后，小龙虾胰蛋白酶和牛胰蛋白酶的解链温度分别升高了27℃和4.5℃。为了探究造成这种分类群特异性的结构特征，我们使天然小龙虾胰蛋白酶与化学合成的SGTI形成复合物并进行结晶。这是节肢动物胰蛋白酶的首个X射线结构，也是迄今为止报道的胰蛋白酶-蛋白质抑制剂复合物的最高分辨率（1.2埃）结构。结构数据表明，除了SGTI的主要结合环（残基P3-P3'）外，SGTI与小龙虾酶之间的相互作用还延伸到了P12-P4和P4'-P5'区域。这部分是由于抑制剂与小龙虾胰蛋白酶结合诱导SGTI结构中P10-P4区域发生了结构变化。通过基于结构的计算比较SGTI-小龙虾胰蛋白酶复合物和SGTI-牛胰蛋白酶复合物发现，SGTI-小龙虾胰蛋白酶复合物在整个结合区域存在显著的相互作用能盈余。与牛胰蛋白酶相比，SGTI与小龙虾胰蛋白酶结合更强且更具特异性的新区域提供了新的抑制剂位点，可用于设计开发高效且特异性的蛋白酶抑制剂以供实际应用。
