Multiple domains of the co-chaperone Hop are important for Hsp70 binding
Multiple domains of the co-chaperone Hop are important for Hsp70 binding
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DOI：
10.1074/jbc.m314130200
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发表时间：
2004-04-16
影响因子：
4.8
通讯作者：
Smith, DF
中科院分区：
文献类型：
--
作者：
Carrigan, PE;Nelson, GM;Smith, DF
Hop/Sti1 共伴侣结合 Hsp70 和 Hsp90。生化和共晶体学研究表明，Hsp70 或 Hsp90 的含有 EEVD 的 C 末端分别与啤酒花四三肽重复结构域之一（TPR1 或 TPR2a）特异性结合。对 Hsp70 和 Hop 进行突变分析是为了更好地表征 Hsp70 和 Hop 结构域 C 末端之间的相互作用。令人惊讶的是，EEVD 的截短加上来自 Hsp70 C 末端的多达 34 个额外氨基酸并没有降低 Hsp70 突变体与 Hop 共免疫沉淀的能力，尽管进一步截短消除了 Hop 结合。正如预期的那样，针对 TPR1 中的羧酸钳位置的跳跃点突变破坏了 Hsp70 结合；然而，TPR2a 或 TPR2b 中的类似点突变在某些情况下也会抑制 Hsp70 结合。使用基于酵母的体内 Hop 功能测定，野生型 Hop 和 TPR2b 突变体可以完全补充 Sti1p 的缺失； TPR1和TPR2a点突变体可以部分恢复活性。通过有限的蛋白水解作用来探测 Hop 和 Hop 突变体的构象。 TPR1突变体以与野生型相似的方式消化；然而，TPR2a 和 TPR2b 突变体均对胰凝乳蛋白酶消化表现出更大的抵抗力。所有点突变体都保留了二聚化的能力，并且没有一个出现严重错误折叠。这些结果对当前 Hop/Hsp70 相互作用模型提出了疑问。
The Hop/Sti1 co-chaperone binds to both Hsp70 and Hsp90. Biochemical and co-crystallographic studies have suggested that the EEVD-containing C terminus of Hsp70 or Hsp90 binds specifically to one of the Hop tetratricopeptide repeat domains, TPR1 or TPR2a, respectively. Mutational analyses of Hsp70 and Hop were undertaken to better characterize interactions between the C terminus of Hsp70 and Hop domains. Surprisingly, truncation of EEVD plus as many as 34 additional amino acids from the Hsp70 C terminus did not reduce the ability of Hsp70 mutants to co-immunoprecipitate with Hop, although further truncation eliminated Hop binding. Hop point mutations targeting a carboxylate clamp position in TPR1 disrupted Hsp70 binding, as was expected; however, similar point mutations in TPR2a or TPR2b also inhibited Hsp70 binding in some settings. Using a yeast-based in vivo assay for Hop function, wild type Hop and TPR2b mutants could fully complement deletion of Sti1p; TPR1 and TPR2a point mutants could partially restore activity. Conformations of Hop and Hop mutants were probed by limited proteolysis. The TPR1 mutant digested in a similar manner to wild type; however, TPR2a and TPR2b mutants each displayed greater resistance to chymotryptic digestion. All point mutants retained an ability to dimerize, and none appeared to be grossly misfolded. These results raise questions about current models for Hop/Hsp70 interaction.