The heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized ATP-binding domain in the carboxyl terminus of the chaperone
The heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized ATP-binding domain in the carboxyl terminus of the chaperone
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DOI：
10.1074/jbc.m003701200
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发表时间：
2000-11-24
影响因子：
4.8
通讯作者：
Neckers, LM
中科院分区：
文献类型：
--
作者：
Marcu, MG;Chadli, A;Neckers, LM
热休克蛋白90(Hsp90)是真核生物中含量最丰富的伴侣蛋白之一，参与类固醇激素受体和蛋白激酶等信号转导分子的折叠和稳定，其氨基末端含有一个非常规的核苷酸结合位点，与细菌DNA旋转酶的ATP结合基序相关。抗肿瘤药物格尔达那霉素和自由基在这个部位特异结合，并诱导依赖于Hsp90的客户蛋白的不稳定。我们最近证实，旋转酶抑制剂novobiocin也可以与Hsp90相互作用，改变伴侣对格尔达霉素和自由基的亲和力，并在体外和体内导致关键的调节Hsp90依赖的激酶包括v-Src、Raf-L和p185(ExbB2)的耗竭。在本研究中，我们使用缺失/突变分析来确定novobiocin与Hsp90的相互作用部位，并且我们证明novobiocin结合部位位于伴侣的羧基末端。令人惊讶的是，这个基序也识别ATP，并且ATP和novobiocin有效地相互竞争结合到Hsp90的这个区域。Novobiocin干扰辅助伴侣Hsc70和p23与Hsp90的结合。这些结果确定了Hsp90上的第二个位点，在那里小分子抑制剂的结合可以显著影响该伴侣的功能，并支持了Hsp90的氨基和羧基末端结构域相互作用调节伴侣活性的假设。
Heat shock protein 90 (Hsp90), one of the most abundant chaperones in eukaryotes, participates in folding and stabilization of signal-transducing molecules including steroid hormone receptors and protein kinases, The amino terminus of Hsp90 contains a non-conventional nucleotide-binding site, related to the ATP-binding motif of bacterial DNA gyrase. The anti-tumor agents geldanamycin and radicicol bind specifically at this site and induce destabilization of Hsp90-dependent client proteins. We recently demonstrated that the gyrase inhibitor novobiocin also interacts with Hsp90, altering the affinity of the chaperone for geldanamycin and radicicol and causing in vitro and in vivo depletion of key regulatory Hsp90-dependent kinases including v-Src, Raf-l, and p185(ExbB2). In the present study we used deletion/mutation analysis to identify the site of interaction of novobiocin with Hsp90, and we demonstrate that the novobiocin-binding site resides in the carboxyl terminus of the chaperone. Surprisingly, this motif also recognizes ATP, and ATP and novobiocin efficiently compete with each other for binding to this region of Hsp90. Novobiocin interferes with association of the cochaperones Hsc70 and p23 with Hsp90. These results identify a second site on Hsp90 where the binding of small molecule inhibitors can significantly impact the function of this chaperone, and they support the hypothesis that both amino- and carboxyl-terminal domains of Hsp90 interact to modulate chaperone activity.