【摘要】 许多重要的生物过程 ,如酶原激活、肽激素合成、病毒蛋白加工和受体成熟 ,均须蛋白质前体加工酶的剪切处理。因此 ,蛋白质前体加工酶可能是一种新药开发的对象。综合利用同源模建技术和序列的进化踪迹分析手段 ,研究了蛋白质前体加工酶furin/kexin与水蛭抑制剂 (eglinC)突变体的相互作用模式 ,阐释furin/kexin各个亚类的底物 /抑制剂特异性的共性和差异性的序列结构基础。在此基础上 ,利用界面再设计策略 (核心算法为异型自洽系综最优化 )进行了furin/kexin抑制剂的理性再设计。分别以模建的水蛭抑制剂 furin ,水蛭素 kex2复合物结构为模板 ,对水蛭抑制剂P1,P2 和P4 位置进行设计 ,计算结果显示这三个位置均是偏好碱性残基 ,与已有的实验结论一致。另外针对furin/kexin各亚类在S′端有较多的特异性残基位置这一特点 ,对抑制剂P′端的残基位置实施改造 ,设计furin和kex2的特异性更高的抑制剂。对于furin ,设计得到的最好突变体是P2 ′Glu P3′Asp P4 ′Arg ;而对于kex2 ,最好的突变体是P2 ′Arg P3′Arg P4 ′Glu ,结构分析显示furin和kex2与相应的水蛭抑制剂突变体形成不同的相互作用模式。这里我们给出了综合利用同源模建技术 ,序列的进化踪迹分析和理性再设计进行酶 抑制剂相互作用研究及抑制剂改?更多还原

【Abstract】 Furin/kexin processing proteases catalyze the proteolysis of large protein precursors involved in many biological processes, such as zymogen activation, peptide hormone synthesis, viral protein processing and receptor maturation, making them potential targets for therapeutic agents. Herein, homology modeling and weighted evolutionary tracing were combined to investigate the interaction mechanism of furin/kex2 with eglin C mutants. The model structures showed that there were many acidic residues in the furin (kex2) binding interface, contributing to specificity for multiple basic residues of their corresponding substrates or inhibitors. Besides, some rational explanations were presented for the different inhibitor/substrate specificity of the furin/kexin members by combining the model structures with results of evolutionary tracing. Based on these analyses, an attempt was made to rationally redesign the eglin C by interface engineering with heterogeneous self consistent ensemble optimization to improve its inhibitory specificity on furin/kex2. With the model complex structures of furin/kex2 and eglin C variants as structural templates, the P 1, P 2 and P 4 of eglin C were redesigned, respectively. The design results show that both furin and kex2 favored basic residues at P 1, P 2 and P 4 in eglin C, in good agreement with the experimental data. The detection of many specific residues in S′ part of furin/kexin sequences made possible designing inhibitors with high specific binding to furin and kex2, respectively. As for furin, the best inhibitor designed was eglin C P 2′Glu P 3′AspP 4′Arg (only these three positions were shown), while the best eglin C variant for kex2 designed was P 2′Arg P 3′Arg P 4′Glu. The structures show that furin and kex2 form distinct interactions with these two eglin C variants. Herein, a strategy was proposed that combine homology modeling, evolutionary tracing and rational interface redesign to investigate enzyme inhibitor interactions and inhibitor engineering. This computational design gives some rational guidance to further experimental inhibitor engineering.更多还原