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...

Previous studies have demonstrated the ATP-dependent formation of a complex containing the heat shock protein hsp90, the unique hsp90 binding protein p23, and one of three high molecular weight immunophilins. In the present study, hsp90 and p23 are shown to form a complex that requires elevated temperature and ATP/ Mg 2؉ . Complex formation is strongly promoted by molybdate and by the nonionic detergent Nonidet P-40. ADP and the benzoquinone ansamycin, geldanamycin, are potent inhibitors of complex formation. The ATP-dependent process alters the state of hsp90, not p23, and influences the ability of hsp90 to bind to phenyl-Sepharose. Conversion of hsp90 to the ATP-bound state lowers its affinity for phenyl-Sepharose. These results show that hsp90 can exist in at least two functional states, one able to bind p23 and the other with a high affinity for hydrophobic resins. A model is presented where these states are dictated by the binding of either ATP or ADP.

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.

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, andXenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes.However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.Numerous recent studies (2,7,8,10,34,49,54) have shown that one of the major heat shock proteins, hsp70, functions in an ATP-dependent manner through transient interactions to mediate folding or unfolding of polypeptide chains. Another major heat shock protein, hsp90, is thought to perhaps also function in some capacity related to folding or protein-protein interactions, but its function(s) remains poorly defined (1). Supporting its potential role in protein folding is a recent demonstration that hsp90 enhances renaturation of some proteins in vitro (52). Perhaps the most widely studied interaction of hsp90 is its identity as a stable component of several unactivated steroid receptor complexes (6, 38, 41). For glucocorticoid receptors, hsp90 binding to receptor is required to maintain high-affinity ligand binding (3,26,40), but other steroid receptors that have been examined do not show this same dependency on hsp90. In all hsp90-nuclear receptor complexes, ligand-dependent activation of the receptor DNA-binding ability is accompanied by dissociation of hsp90 (14,17,31,43), and it appears likely that one hsp90 function is to repress DNA binding by receptor.Steroid receptor-hsp9O interactions provide a model for understanding hsp90 function, but exploiting this model has been hindered by the inability to reversibly assemble receptor-hsp90 complexes in vitro. This drawback was recently overcome by establishing certain physicochemical conditions that permit the use of rabbit reticulocyte...

The highly coordinated interactions of several molecular chaperones, including hsp70 and hsp90, are required for the folding and conformational regulation of a variety of proteins in eukaryotic cells, such as steroid hormone receptors and many other signal transduction regulators. The protein called Hop serves as an adaptor protein for hsp70 and hsp90 and is thought to optimize their functional cooperation. Here we characterize the assembly of the hsp70-Hop-hsp90 complex and reveal interactions that cause conformational changes between the proteins in the complex. We found that hsp40 plays an integral role in the assembly by enhancing the binding of hsp70 to the Hop complex. This is accomplished by stimulating the conversion of hsp70-ATP to hsp70-ADP, the hsp70 conformation favored for Hop binding. The hsp70-Hop-hsp90 complex is highly dynamic, as has been observed previously for hsp90 in its interaction with client proteins. Nonetheless, hsp90 binds with high affinity to Hop (K d ‫؍‬ 90 nM), and this binding is not affected by hsp70. hsp70 binds with lower affinity to Hop (K d ‫؍‬ 1.3 M) on its own, but this affinity is increased (K d ‫؍‬ 250 nM) in the presence of hsp90. hsp90 also reduces the number of hsp70 binding sites on the Hop dimer from two sites in the absence of hsp90 to one site in its presence. Hop can inhibit the ATP binding and p23 binding activity of hsp90, yet this can be reversed if hsp70 is present in the complex. Taken together, our results suggest that the assembly of hsp70-Hop-hsp90 complexes is selective and influences the conformational state of each protein.