Interaction of E. coli Hsp90 with DnaJ Involves the DnaJ Binding Region of DnaK
Interaction of E. coli Hsp90 with DnaJ Involves the DnaJ Binding Region of DnaK
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DOI：
10.1016/j.jmb.2016.12.014
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发表时间：
2017-03-24
影响因子：
5.6
通讯作者：
Wickner, Sue
中科院分区：
文献类型：
--
作者：
Kravats, Andrea N.;Doyle, Shannon M.;Wickner, Sue
热休克蛋白90（Hsp90）是一种广泛存在于原核生物和真核生物中的保守分子伴侣，参与ATP依赖的蛋白质重构。它与Hsp70和Hsp70辅助分子伴侣、Hsp40（J蛋白）和核苷酸交换因子一起发挥功能。在大肠杆菌中，Hsp90（Ec）和Hsp70（DnaK）之间的功能协作需要两种分子伴侣直接相互作用。我们使用分子对接来模拟Hsp90（Ec）和DnaK的相互作用。排名靠前的对接模型预测DnaK的核苷酸结合结构域（NBD）中的区域与Hsp90Ec的中间结构域中的区域相互作用。然后，我们在模型建议的DnaK残基中制备了取代突变体，以与Hsp90（EC）相互作用。测试的12个突变体中，11个是有缺陷的或部分缺陷的能力，在体内与热休克蛋白90（Ec）在细菌双杂交试验和在体外的生物层干涉测定相互作用。这些DnaK突变体在与Hsp90 E的蛋白质重塑中协同发挥作用的能力也有缺陷，但保留了与DnaK辅伴侣作用的能力。综上所述，这些结果表明DnaK的NBD中的特定区域参与与Hsp90（Ec）的相互作用，并且这种相互作用在功能上是重要的。此外，我们发现Hsp90（Ec）结合所必需的DnaK区域包括也参与J蛋白结合的残基，表明DnaK、DnaK辅伴侣蛋白和Hsp9（OE）c之间存在功能性相互作用。爱思唯尔有限公司出版
The 90-kDa heat shock protein (Hsp90) is a widely conserved and ubiquitous molecular chaperone that participates in ATP-dependent protein remodeling in both eukaryotes and prokaryotes. It functions in conjunction with Hsp70 and the Hsp70 cochaperones, an Hsp40 (J-protein) and a nucleotide exchange factor. In Escherichia coli, the functional collaboration between Hsp90(Ec) and Hsp70, DnaK, requires that the two chaperones directly interact. We used molecular docking to model the interaction of Hsp90(Ec) and DnaK. The top-ranked docked model predicted that a region in the nucleotide-binding domain (NBD) of DnaK interacted with a region in the middle domain of Hsp90Ec. We then made substitution mutants in DnaK residues suggested by the model to interact with Hsp90(Ec). Of the 12 mutants tested, 11 were defective or partially defective in their ability to interact with Hsp90(Ec) in vivo in a bacterial two-hybrid assay and in vitro in a bio-layer interferometry assay. These DnaK mutants were also defective in their ability to function collaboratively in protein remodeling with Hsp90E, but retained the ability to act with DnaK cochaperones. Taken together, these results suggest that a specific region in the NBD of DnaK is involved in the interaction with Hsp90(Ec), and this interaction is functionally important. Moreover, the region of DnaK that we found to be necessary for Hsp90(Ec) binding includes residues that are also involved in J-protein binding, suggesting a functional interplay among DnaK, DnaK cochaperones, and Hsp9(0E)c. Published by Elsevier Ltd.