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.

Heat shock protein (hsp)90 functions in a complex chaperoning pathway where its activity is modulated by ATP and by interaction with several co-chaperones. One co-chaperone, p23, binds selectively to the ATP-bound state of hsp90. However, the isolated ATP-binding domain of hsp90 does not bind p23. In an effort to identify the p23-binding domain, we have constructed a series of hsp90 deletion mutants fused with glutathione-S-transferase (GST). Full-length GST-hsp90 is able to bind p23, and also, to chaperone assembly of progesterone receptor complexes. Truncations from the C terminus of GST-hsp90 reveal a C-terminal boundary for the p23-binding domain at approximately residue 490. This fragment contains, in order, the ATP-binding domain, a highly charged region, and 203 residues beyond the charged region. p23 binding is unaffected by deletion of the charged region, indicating that two noncontiguous regions of hsp90 are involved in p23 binding. These regions are only effective when hsp90 is in a dimeric state as shown by loss of p23 binding upon removal of GST or as shown by use of FK506-binding protein12-hsp90 constructs that form dimers and bind p23 only in the presence of a bivalent drug. Thus, p23 binding requires an hsp90 dimer with close proximity between N-terminal regions of hsp90 and a conformation specified by ATP.H eat shock protein (hsp)90 is an abundant and ubiquitous molecular chaperone that is required to assist the conformational maturation of specific targets involved in many key functions of the cell such as cell-cycle regulation and signal transduction. Among hsp90 targets are several protein kinases such as v-Src, Weel, and c-Raf, transcriptional regulators such as p53 and steroid receptors, and two polymerases: that of hepatitis B virus and telomerase (for review, see refs. 1-4). To date, the best known maturation process driven by hsp90 is the assembly of steroid receptors into a high-affinity hormone-binding conformation. This complex, multistep process occurs in an ATPdependent manner and involves several other chaperones and co-chaperones (4). Three dynamic steps have been observed in this assembly process (5-7). The receptor initially associates with hsp70, assisted by hsp40. This association develops into an intermediate complex in which the co-chaperone, hsp organizing protein (Hop), associates with both hsp70 on the receptor and hsp90, while the protein Hip binds to hsp70. As the receptor complex matures, hsp90 bound to the receptor interacts with p23 and any one of four tetratricopeptide repeat-containing proteins, FK506-binding protein (FKBP)52, FKBP51, cyclophilin-40, or phosphoprotein phosphatase 5. The details of the interactions between these partner proteins are poorly understood and the mechanisms driving the transitions from one complex to another are still unresolved.hsp90 is a dimeric protein with multiple domains. Several proteins involved in the hsp90 chaperone pathway possess structurally related tetratricopeptide repeat motifs that mediated their interaction with hsp90 a...

Hsp90, a molecular chaperone required for the functioning of glucocorticosteroid receptor (GR), ensures, by direct interaction, the conformational competence of the steroid-binding pocket. In addition to having this positive function, Hsp90 maintains steroid receptors in an inactive form in the absence of hormone. However, neither the participation of Hsp90 once the pathway has been activated by the ligand nor the importance of increased Hsp90 levels in determining the amplitude of the response has ever been assessed directly. Here, by increasing the Hsp90͞GR ratio in the nuclear compartment, we found an attenuation of the response to glucocorticosteroids that was not due to a nonspecific or toxic effect of the Hsp90 modified by nuclear targeting. Since this negative effect was more pronounced at high levels of hormone, when receptor and Hsp90 are maximally dissociated, the possibility of an interaction between Hsp90 and GR, already activated to a DNA-binding form, was directly investigated. Indeed GR, after in vivo activation by ligand, was still able to reassociate with Hsp90, suggesting that this interaction plays a role in vivo, possibly in receptor recycling. Moreover, the GR binding to its DNA response element was inhibited by an excess of Hsp90, pointing to a function of Hsp90 in the nuclear compartment. It is thus proposed that an increased Hsp90͞GR ratio inf luences the responsiveness to ligand at a step that is after receptor activation. This increased ratio may be of pathophysiological relevance in the different circumstances that lead to an elevated level of nuclear Hsp90.

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