Many functions of the chaperone, heat shock protein 90 (hsp90), are inhibited by the drug geldanamycin that specifically binds hsp90. We have studied an amino-terminal domain of hsp90 whose crystal structure has recently been solved and determined to contain a geldanamycin-binding site. We demonstrate that, in solution, drug binding is exclusive to this domain. This domain also binds ATP linked to Sepharose through the ␥-phosphate. Binding is specific for ATP and ADP and is inhibited by geldanamycin. Mutation of four glycine residues within two proposed ATP binding motifs diminishes both geldanamycin binding and the ATP-dependent conversion of hsp90 to a conformation capable of binding the co-chaperone p23. Since p23 binding requires regions outside the 1-221 domain of hsp90, these results indicate a common site for nucleotides and geldanamycin that regulates the conformation of other hsp90 domains.Heat shock protein 90 (hsp90) 1 is a cellular chaperone that participates in multiple signal transduction pathways. Recent studies have demonstrated a requirement for hsp90, or grp94, its homolog in the endoplasmic reticulum, for the proper function of 1) the mitogen-activated protein kinase pathway (1-6); 2) activity of several tyrosine kinases (Refs. 7-9 and references therein); 3) activity of several transcription factors, including p53 (10), retinoid receptors (11), steroid and aryl hydrocarbon receptors (Refs. 12 and 13 and references therein), and hypoxia-inducible factor ␣ (14); 4) activity of the cyclin-dependent kinase CDK4 (15) and the cell cycle-associated Wee1 tyrosine kinase (16); and even 5) activity of hepatitis B virus reverse transcriptase (17). Additionally, hsp90 has been shown to participate in the refolding of certain misfolded proteins (18 -20). hsp90 comprises the core of several multi-molecular chaperone complexes that interact with proteins at different stages of their maturation. The ability of hsp90 to participate in the assembly of multiple higher order chaperone complexes no doubt contributes to its involvement in diverse cellular pathways, although those factors regulating such participation remain unclear.Until recently, yeast in which hsp90 is either mutated or conditionally suppressed has served as the only means by which to study the many functions of this chaperone in the cell. The recent observation that a class of drugs known as benzoquinone ansamycins, including herbimycin A and geldanamycin (GA), specifically bind and inhibit hsp90 and grp94 has provided a new tool for functional studies of these chaperones (9, 21). Indeed, a study of structure-activity relationships has demonstrated a high correlation between the biologic effects of the benzoquinone ansamycins and their ability to bind hsp90 (22). These drugs have also been shown to possess anti-tumor activity in preclinical models, identifying the hsp90 chaperone family as a novel target for anticancer drug development (23).For these reasons, it is of much interest to characterize the drug binding site in hsp90, both to underst...

Cytosolic Raf-1 exists in a high molecular weight complex with the heat shock protein Hsp90, the purpose of which is unknown. The benzoquinone ansamycin, geldanamycin, specifically binds to Hsp90 and disrupts certain multimolecular complexes containing this protein.Using this drug, we are able to demonstrate rapid dissociation of both Raf-1-Hsp90 and Raf-1-Ras multimolecular complexes, concomitant with a markedly decreased halflife of the Raf-1 protein. Continued disruption of the Raf-1-Hsp90 complex results in apparent loss of Raf-1 protein from the cell, although Raf-1 synthesis is actually increased. Prevention of Raf-1-Hsp90 complex formation interferes with trafficking of newly synthesized Raf-1 from cytosol to plasma membrane. These data indicate that association with Hsp90 is essential for both Raf-1 protein stability and its proper localization in the cell.

17-AG shares the important biologic features of its parent compound GA. Since 17-AG has a better toxicity profile than GA, it is an interesting candidate benzoquinone ansamycin for clinical development.

The molecular chaperone Hsp90 plays an essential role in the folding and function of important cellular proteins including steroid hormone receptors, protein kinases and proteins controlling the cell cycle and apoptosis. A 15 A deep pocket region in the N-terminal domain of Hsp90 serves as an ATP/ADP-binding site and has also been shown to bind geldanamycin, the only specific inhibitor of Hsp90 function described to date. We now show that radicicol, a macrocyclic antifungal structurally unrelated to geldanamycin, also specifically binds to Hsp90. Moreover, radicicol competes with geldanamycin for binding to the N-terminal domain of the chaperone, expressed either by in vitro translation or as a purified protein, suggesting that radicicol shares the geldanamycin binding site. Radicicol, as does geldanamycin, also inhibits the binding of the accessory protein p23 to Hsp90, and interferes with assembly of the mature progesterone receptor complex. Radicicol does not deplete cells of Hsp90, but rather increases synthesis as well as the steady-state level of this protein, similar to a stress response. Finally, radicicol depletes SKBR3 cells of p185erbB2, Raf-1 and mutant p53, similar to geldanamycin. Radicicol thus represents a structurally unique antibiotic, and the first non-benzoquinone ansamycin, capable of binding to Hsp90 and interfering with its function.