鲢鱼肌原纤维结合型丝氨酸蛋白酶的结构建模、内源抑制剂对接及其钙结合机制

Structure-modeling,Endogenous Inhibitor-docking and Calcium-binding Mechanism of Myofibril-bound Serine Proteinase from Silver Carp(Hypophthalmichthys molitrix)

【目的】分析鲢(Hypophthalmichthys molitrix)肌原纤维结合型丝氨酸蛋白酶(Myofibril-bound serine proteinase,MBSP)结构的基础特性。【方法】使用AlphaFold2对鲢MBSP进行结构建模,通过保守性分析、静电势计算、分子对接等手段探讨鲢鱼MBSP的结构特征,并借助分子动力学模拟研究钙结合对其结构的影响。【结果与结论】鲢鱼MBSP的活性酶形式含222个残基,有胰蛋白酶样丝氨酸蛋白酶的典型结构特征,但表面充斥着更多赖氨酸,使其表面“粗糙”且利于离子键形成。62Asn及61Glu是其关键的钙结合位点,钙结合导致的结构变动可传至C端,40His、自溶环、氧阴离子稳定环等关键区域的柔性变化是钙激活机制中的重要部分。葡萄糖-6-磷酸异构酶二聚体上的10Arg及17Lys是参与鲢鱼MBSP底物结合竞争的潜在关键位点。除催化活性口袋外,潜在的变构位点亦是MBSP抑制剂设计的可选出发点。

【Objective】To study the basic characteristics of the structure of myofibril-bound serine proteinase(MBSP)in silver carp(Hypophthalmichthys molitrix).【Method】AlphaFold2 was used for structure-modeling for MBSP.Structural features of MBSP from silver carp were investigated by means of conservation analysis,calculation of electrostatic potential and molecular docking.Then,molecular dynamic simulation was used to investigate the effect of calcium binding on the enzyme structure.【Result and Conclusion】Composed of 222 residues,the activated MBSP typically shows structural characteristics of trypsin-like serine proteinase.But there are more lysines on the surface of MBSP,inducing a"rough"surface and nice opportunity for the formation of ionic bonds.62Asn and 61Glu are the crucial calcium-binding sites.Structural variations caused by calcium binding can be transmitted to the C-terminus.Changes in the flexibility of vital regions(e.g.,40His,the autolysis loop,the oxyanion stabilizing loop)possibly played a major role in the calcium activation.10Arg and 17Lys of glucose-6-phosphate isomerase dimer are likely to compete for binding sites with the substrates of MBSP.Apart from the catalytic pocket,the potential allosteric sites are also optional starting points for MBSP inhibitor design.