The Heat Shock Protein 90 Antagonist Novobiocin Interacts with a Previously Unrecognized ATP-binding Domain in the Carboxyl Terminus of the Chaperone

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 p185ErbB2 . 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.

Comparative analysis of the ATP‐binding sites of Hsp90 by nucleotide affinity cleavage: a distinct nucleotide specificity of the C‐terminal ATP‐binding site

“…c-Phosphate-linked ATP-Sepharose binding has been used as the first biochemical assay for the unambiguous identification of Hsp90 as an ATP-binding protein by Grenert et al [9]. Though C-terminal fragments of Hsp90 also bound to the resin [19], and we demonstrated that Hsp90 was able to bind in the presence of a saturating concentration of the N-terminal inhibitor, GA [20], others could not detect binding under these circumstances [29]. We were intrigued by this apparent contradiction, and made an additional attempt to resolve the discrepancy.…”

“…Recent communications have reported a second ATPbinding site in the C-terminal domain of Hsp90 [19][20][21]. Our studies demonstrated that the C-terminal nucleotide binding site becomes accessible only after the occupancy of the N-terminal site and is sensitive to cisplatin [20].…”

“…They showed a competition with soluble ADP and ATP, but not with GTP up to 5 mM [9]. On the contrary, recent experiments on N-terminally truncated Hsp90 constructs suggested that GTP, indeed, may bind to the C-terminal domain [19]. Using different fluorescent ATP analogs, including N 6 -etheno-ATP, Scheibel et al [6] could not detect a high affinity ATP-binding to Hsp90.…”

“…Since the available data in the literature is rather sporadic, and previous experiments obviously could not take into account the existence of the second ATP-binding site on Hsp90, which has been uncovered just recently [19][20][21], in the present study we undertook a systematic and comparative analysis of the nucleotide specificity of both the N-terminal and C-terminal Hsp90 nucleotide binding sites. In this study we demonstrate that oxidative nucleotide affinity cleavage is a useful technique to characterize the nucleotide binding sites of Hsp90.…”

Conformational dynamics of the molecular chaperone Hsp90

“…Novobiocin is a small molecule inhibitor that binds to the CTD of Hsp90 (Marcu et al, 2000a;Marcu et al, 2000b;Soti et al, 2002). Like GA and radicicol, novobiocin inhibits the formation of Hsp90 complexes with client or p23, but unlike GA it also reduces the affinity of Hsp90 for TPR-containing co-chaperones that bind to the CTD (Allan et al, 2006;Yun et al, 2004).…”