Mechanistic Insight Into the Interaction Between Helicobacter pylori Urease Subunit α and Its Molecular Chaperone Hsp60
Mechanistic Insight Into the Interaction Between Helicobacter pylori Urease Subunit α and Its Molecular Chaperone Hsp60
复制标题
幽门螺杆菌脲酶α亚基与其分子伴侣Hsp60相互作用的机制洞察
DOI：
10.3389/fmicb.2019.00153
复制
发表时间：
2019
影响因子：
5.2
通讯作者：
Xiaofei Ji
中科院分区：
文献类型：
--
作者：
Huilin Zhao;Yulong Wu;Zheng Xu;Ran Ma;Yunfei Ding;Xuelian Bai;Qianyu Rong;Ying Zhang;Boqing Li;Xiaofei Ji
幽门螺杆菌是多种胃十二指肠疾病的病原体。尿素酶和热休克蛋白60作为其主要致病因子，在H.幽门。以往的研究表明，脲酶与Hsp 60之间存在着密切的关系，这意味着Hsp 60可能在脲酶的稳定和组装过程中起着分子伴侣的作用。然而，这两种蛋白质如何相互作用仍不清楚。研究了Hsp 60对H.首先检测pylori裂解物以确认尿素酶和Hsp 60之间的相互作用。Pull-down实验进一步表明Hsp 60可与UreA亚基结合，但不与UreB结合。然后，利用I-TASSER软件对Hsp 60的三维结构进行了建模，通过分子对接模拟了Hsp 60与UreA的结合复合物。结果表明，UreA与Hsp 60的空腔完全吻合。分析所得到的模型表明，至少有七个残基的尿素，位于两个接口，参与相互作用。通过表面等离子体共振（SPR）实验，这些潜在残基的定点诱变显示与Hsp 60的亲和力比野生型UreA降低，并且D 68似乎在亲和力中具有重要作用。进一步的分析还表明，E25和K26的突变导致了比野生型UreA更快的缔合和解离，这意味着它们具有稳定相互作用复合物的作用。这些亲和力比较表明，分子对接预测的接口是可信的。我们的研究表明Hsp 60和尿素酶之间存在直接的相互作用，并揭示了相互作用的结合界面和关键残基。这些结果为Hsp 60对尿素酶的伴侣活性提供了进一步的证据，并为更好地理解Hsp 60中尿素酶的成熟机制奠定了基础。幽门。
Helicobacter pylori is the etiologic agent in a variety of gastroduodenal diseases. As its key pathogenic factors, both urease and Hsp60 play important roles in the pathogenesis of H. pylori. Previous studies have suggested that there is close relationship between urease and Hsp60, which implied that Hsp60 may act as a chaperone in urease stabilization and assembly. However, how these two proteins interact remains unclear. In this study, the impact of Hsp60 on urease activity of H. pylori lysate was first detected to confirm the interaction between urease and Hsp60. Pull-down assays further indicated that Hsp60 could bind to UreA subunit but not UreB. Then, the 3D structure of Hsp60 was modeled using I-TASSER to simulate the binding complex with UreA by molecular docking. The results showed that UreA is a perfect fit for the cavity of Hsp60. Analysis of the resulting model demonstrated that at least seven residues of UreA, located on two interfaces, participate in the interaction. Site-directed mutagenesis of these potential residues showed reduced affinity with Hsp60 than the wild type UreA through surface plasmon resonance (SPR) experiments, and D68 appears to have an important role in the affinity. Further analysis also showed that mutation of E25 and K26 caused a more rapid association and dissociation than with wild UreA, implying that they have roles in stabilizing the interaction complex. These affinity comparisons suggested that the interfaces predicted by molecular docking are credible. Our study indicated a direct interaction between Hsp60 and urease and revealed the binding interfaces and key residues involved in the interaction. These results provide further evidence for the chaperone activity of Hsp60 toward urease and lay a foundation to better understand the maturation mechanism of urease in H. pylori.