Kim Soon-Young, Sharma Suveena, Hoskins Joel R, Wickner Sue
Laboratory of Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892-4264, USA.
J Biol Chem. 2002 Nov 22;277(47):44778-83. doi: 10.1074/jbc.M206176200. Epub 2002 Sep 16.
DnaK, the Hsp70 chaperone of Escherichia coli interacts with protein substrates in an ATP-dependent manner, in conjunction with DnaJ and GrpE co-chaperones, to carry out protein folding, protein remodeling, and assembly and disassembly of multisubunit protein complexes. To understand how DnaJ targets specific proteins for recognition by the DnaK chaperone system, we investigated the interaction of DnaJ and DnaK with a known natural substrate, bacteriophage P1 RepA protein. By characterizing RepA deletion derivatives, we found that DnaJ interacts with a region of RepA located between amino acids 180 and 200 of the 286-amino acid protein. A peptide corresponding to amino acids 180-195 inhibited the interaction of RepA and DnaJ. Two site-directed RepA mutants with alanine substitutions in this region were about 4-fold less efficiently activated for oriP1 DNA binding by DnaJ and DnaK than wild type RepA. We also identified by deletion analysis a site in RepA, in the region of amino acids 35-49, which interacts with DnaK. An alanine substitution mutant in amino acids 36-39 was constructed and found defective in activation by DnaJ and DnaK. Taken together the results suggest that DnaJ and DnaK interact with separate sites on RepA.
DnaK是大肠杆菌的热休克蛋白70（Hsp70）伴侣蛋白，它与辅伴侣蛋白DnaJ和GrpE协同作用，以ATP依赖的方式与蛋白质底物相互作用，从而进行蛋白质折叠、蛋白质重塑以及多亚基蛋白质复合物的组装和拆卸。为了了解DnaJ如何将特定蛋白质靶向至DnaK伴侣蛋白系统进行识别，我们研究了DnaJ和DnaK与一种已知的天然底物——噬菌体P1 RepA蛋白之间的相互作用。通过对RepA缺失衍生物进行表征，我们发现DnaJ与RepA位于该286个氨基酸的蛋白质的第180至200位氨基酸之间的区域相互作用。一段对应于第180 - 195位氨基酸的肽抑制了RepA与DnaJ的相互作用。在该区域具有丙氨酸替代的两个定点RepA突变体，与野生型RepA相比，被DnaJ和DnaK激活用于oriP1 DNA结合的效率低约4倍。我们还通过缺失分析确定了RepA中位于第35至49位氨基酸区域的一个与DnaK相互作用的位点。构建了一个第36 - 39位氨基酸的丙氨酸替代突变体，发现其在被DnaJ和DnaK激活方面存在缺陷。综合这些结果表明，DnaJ和DnaK与RepA上的不同位点相互作用。
