M.G. Catelli

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.

This work was initiated to determine if a specific region of the glucocorticoid receptor determines the formation of the inactive (i.e. non-DNA-binding) 9 S form of the receptor recovered in cytosol preparations. It is known that the murine glucocorticoid receptor of the nti phenotype, which consists of only the carboxyl-terminal 40-kDa peptide containing the DNA-binding and steroid-binding domains separated by a short linker region, is recovered in hypotonic lysates as a 9 S heteromeric complex (Gehring, U., and Arndt, H. (1985) FEBS Lett. 179, 138-142). To further localize the domain required for formation of the 9 S complex, we have determined the sedimentation coefficients of receptors produced in COS-7 cells transfected with several mutants of the human glucocorticoid receptor gene. Deletion of the DNA-binding domain results in a 9 S complex that is somewhat less stable than the wild type receptor during sucrose gradient centrifugation. Deletion of the linker region yields a molybdate-stabilized 9 S complex, but deletion of the entire steroid-binding domain or internal deletion of the amino-terminal two-thirds of this domain yields receptors that are constitutive transcriptional activators and are present in cytosol only in the 4 S form. Taken together, these observations demonstrate that the steroid-binding domain contains the features required for formation of the 9 S heteromeric complex, and they are consistent with the proposal that the steroid-binding domain normally represses receptor function.

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.

The presence of a nucleotide binding site on hsp90 was very controversial until x-ray structure of the hsp90 N-terminal domain, showing a nonconventional nucleotide binding site, appeared. A recent study suggested that the hsp90 C-terminal domain also binds ATP (Marcu, M. G., Chadli, A., Bouhouche, I., Catelli, M. G., and Neckers, L. M. (2000) J. Biol. Chem. 275, 37181–37186). In this paper, the interactions of ATP with hsp90 and N-terminal and C-terminal and that hsp90 a site on the C-terminal of the The this domain of hsp90 and and a with the binding on the the structure with a the C-terminal of that this the C-terminal also and C-terminal of The presence of a nucleotide binding site on hsp90 was very controversial until x-ray structure of the hsp90 N-terminal domain, showing a nonconventional nucleotide binding site, appeared. A recent study suggested that the hsp90 C-terminal domain also binds ATP (Marcu, M. G., Chadli, A., Bouhouche, I., Catelli, M. G., and Neckers, L. M. (2000) J. Biol. Chem. 275, 37181–37186). In this paper, the interactions of ATP with hsp90 and N-terminal and C-terminal and that hsp90 a site on the C-terminal of the The this domain of hsp90 and and a with the binding on the the structure with a the C-terminal of that this the C-terminal also and C-terminal of and and of the of and the of of of of of the and and also of the the of hsp90 with a of a and that hsp90 of N-terminal and C-terminal a J. of the N-terminal domain of and hsp90 the of the binding site, the M. J. Biol. Chem. site the of the J. J. Biol. Chem. the site, the N-terminal domain hsp90 J. J. Biol. Chem. and a site the binding of J. J. Biol. Chem. In the the structure of the C-terminal domain of hsp90 domain a site, the N-terminal J. the C-terminal Biol. M. J. J. J. and J. of The of the of and M. J. ATP binding and that the hsp90 and the C-terminal of hsp90 the nucleotide the J. M. J. J. Biol. Chem. J. J. J. Biol. a site hsp90 was suggested the of J. Biol. 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