Abstract
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-1, and p185(ErbB2). 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 co-chaperones 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.
Highlights
The abundant molecular chaperone Hsp90 mediates the function of many key regulatory proteins of eukaryotic cells, including steroid receptors [1,2,3], mutated p53 [4], and a number of tyrosine and serine/threonine kinases, among which are members of the Src family [5], p185ErbB2 [6], cyclin-dependent kinases Cdk4 and Cdk6 [7], and Raf-1 [8, 9]
We recently reported that novobiocin, an antibiotic previously shown to bind adjacent to the ATP-binding site of bacterial gyrase B and to interfere with nucleotide binding [24], was able to interact with Hsp90, albeit with lower affinity than with gyrase B, and to disrupt the chaperone activity of Hsp90 in a manner similar to GA and radicicol [25]
The current data, together with our previous study [25], emphasize the functional significance of novobiocin binding to Hsp90, support the possibility that the conformation of Hsp90’s amino terminus can influence the behavior of the carboxyl-terminal portion of the molecule, and suggest that a previously unrecognized second nucleotide-binding domain exists in the carboxyl terminus of Hsp90
Summary
The abundant molecular chaperone Hsp mediates the function of many key regulatory proteins of eukaryotic cells, including steroid receptors [1,2,3], mutated p53 [4], and a number of tyrosine and serine/threonine kinases, among which are members of the Src family [5], p185ErbB2 [6], cyclin-dependent kinases Cdk and Cdk6 [7], and Raf-1 [8, 9]. X-ray crystallographic and biochemical studies have demonstrated that the natural products geldanamycin (GA) and radicicol both interact with the amino-terminal nucleotide-binding site on Hsp90 [16, 17, 19, 20], leading to alterations in the conformation and function of the protein. In contrast to the amino terminus, the crystal structure of the carboxyl-terminal region of Hsp remains undetermined, and biochemical characterization of this portion of the chaperone suggests that a complex interaction between this and the amino-terminal domain is a critical regulatory component of chaperone function [14, 21,22,23]. The current data, together with our previous study [25], emphasize the functional significance of novobiocin binding to Hsp, support the possibility that the conformation of Hsp90’s amino terminus can influence the behavior of the carboxyl-terminal portion of the molecule, and suggest that a previously unrecognized second nucleotide-binding domain exists in the carboxyl terminus of Hsp
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