分子伴侣幽门螺杆菌Hsp60与UreB的相互作用分析

Analysis of interaction between Helicobacter pylori UreB and its chaperone Hsp60

目的检测并分析幽门螺杆菌(Hp)尿素酶β亚基(UreB)与Hsp60之间的相互作用。方法克隆Hp 26695的尿素酶β亚基基因(UreB)融合GST标签和Hsp60基因融合His标签,分别在大肠埃希菌中进行异源表达。提取两种蛋白,采用pull-down方法检测两者间的相互作用。利用Modeller 9v2软件,以E.coli的GroEL为基础模建Hp Hsp60的三维结构,再与已知的UreB结构利用AutoDock 4.2软件进行分子共模拟,分析二者的相互作用面和关键氨基酸。结果构建了Hp Hsp60和尿素酶β亚基(UreB)的大肠埃希菌异源表达体系并获得纯化蛋白;Pull-down试验显示部分未融合GST标签的Hsp60出现在GST-UreB的洗脱液中,表明Hsp60与GST-UreB之间存在相互作用;以E.coli的GroEL为基础模建了Hp Hsp60的三维结构,利用docking技术构建UreB与Hp Hsp60的相互作用模型,发现其主要的相互作用面位于Hsp60的α9与UreB的α2之间,其可能形成氢键的关键位点为UreB的α2上的T147氨基酸与Hsp60的α9上的E237、K238。结论 Hp Hsp60能直接与尿素酶β亚基(UreB)相互作用,Hsp60可能通过这种相互作用在UreB成熟过程中发挥分子伴侣功能。这为阐明Hp尿素酶的组装与成熟机制奠定了基础。

Objective To detect and analyze the interaction between Hsp60 and UreB to substantiate the contention that Hsp60 acts as a chaperone of UreB in Helicobacter pylori. Methods The UreB gene from H.pylori strain 26695 was tagged with GST and the Hsp60 gene was tagged with His,and the two genes were cloned.GST-tagged UreB and Histagged Hsp60 were expressed in E.coli,and the two recombinant proteins were purified using affinity chromatography.Then the interaction between two proteins was analyzed using apull-down assay.To analyze the interaction between Hsp60 and UreB at the molecular level,Hsp60 was modeled based on the structure of E.coli GroEL using Modeller 9v2 software.The structure of UreB was downloaded from PDB database.AutoDock 4.2software was used to model the interaction between these two molecules. Results Systems for heterologous expression of Hsp60 and UreB were successfully constructed.The pull-down assay revealed that a portion of Hsp60 that was not tagged with GST appeared in the eluent.This result suggested that there is direct interaction between Hsp60 and UreB.Based on E.coli GroEL,the 3D structure of H.pylori Hsp60 was modeled and docked into UreB.This model revealed that the major surface of interaction involved anα9helix of Hsp60 and anα2helix of UreB.The key amino acids involved in this interaction may be T147 on theα2helix of UreB and E237/K238 on theα9helix of Hsp60. Conclusion There is direct interaction between Hsp60 and UreB,which implies that Hsp60 can act as a chaperone during the maturation of UreB.This result lays the foundation for ascertaining the mechanism of assembly and maturation of urease in H.pylori.