The Hsp90 chaperone is required for the activation of several families of eukaryotic protein kinases and nuclear hormone receptors, many of which are protooncogenic and play a prominent role in cancer. The geldanamycin antibiotic has antiproliferative and antitumor effects, as it binds to Hsp90, inhibits the Hsp90-mediated conformational maturation/refolding reaction, and results in the degradation of Hsp90 substrates. The structure of the geldanamycin-binding domain of Hsp90 (residues 9-232) reveals a pronounced pocket, 15 A deep, that is highly conserved across species. Geldanamycin binds inside this pocket, adopting a compact structure similar to that of a polypeptide chain in a turn conformation. This, and the pocket's similarity to substrate-binding sites, suggest that the pocket binds a portion of the polypeptide substrate and participates in the conformational maturation/refolding reaction.

Bag (Bcl2-associated athanogene) domains occur in a class of cofactors of the eukaryotic chaperone 70-kilodalton heat shock protein (Hsp70) family. Binding of the Bag domain to the Hsp70 adenosine triphosphatase (ATPase) domain promotes adenosine 5'-triphosphate-dependent release of substrate from Hsp70 in vitro. In a 1.9 angstrom crystal structure of a complex with the ATPase of the 70-kilodalton heat shock cognate protein (Hsc70), the Bag domain forms a three-helix bundle, inducing a conformational switch in the ATPase that is incompatible with nucleotide binding. The same switch is observed in the bacterial Hsp70 homolog DnaK upon binding of the structurally unrelated nucleotide exchange factor GrpE. Thus, functional convergence has allowed proteins with different architectures to trigger a conserved conformational shift in Hsp70 that leads to nucleotide exchange.

The role of the abundant stress protein Hsp90 in protecting cells against stress-induced damage is not well understood. The recent discovery that a class of ansamycin antibiotics bind specifically to Hsp90 allowed us to address this problem from a new angle. We find that mammalian Hsp90, in cooperation with Hsp70, p60, and other factors, mediates the ATP-dependent refolding of heatdenatured proteins, such as firef ly luciferase. Failure to refold results in proteolysis. The ansamycins inhibit refolding, both in vivo and in a cell extract, by preventing normal dissociation of Hsp90 from luciferase, causing its enhanced degradation. This mechanism also explains the ansamycin-induced proteolysis of several protooncogenic protein kinases, such as Raf-1, which interact with Hsp90. We propose that Hsp90 is part of a quality control system that facilitates protein refolding or degradation during recovery from stress. This function is used by a limited set of signal transduction molecules for their folding and regulation under nonstress conditions. The ansamycins shift the mode of Hsp90 from refolding to degradation, and this effect is probably amplified for specific Hsp90 substrates.Exposure of prokaryotic and eukaryotic cells to heat and other stresses induces several classes of highly conserved stress proteins, including the members of the Hsp70, Hsp60, and Hsp90 families (1-3). These proteins are generally thought to act as molecular chaperones in preventing the aggregation of nonnative polypeptides and in aiding their correct folding. Although significant progress has been made in understanding the chaperone mechanisms in de novo protein folding, surprisingly little is known about the role of chaperones under stress conditions. This lack of knowledge is particularly apparent for the Hsp90s, the most abundant constitutively expressed stress proteins in the eukaryotic cytosol. Although Hsp90 can prevent protein aggregation in vitro (4-6) and is required for the survival of yeast at elevated temperature (7), its actual role in protein refolding and repair under stress has remained elusive (2, 8). Instead, current thinking views Hsp90 as part of a specific chaperone system for the conformational maturation and regulation of signal transduction molecules, such as several potentially oncogenic protein kinases and the nuclear receptors of steroid hormones (8-11). In the mammalian cytosol, these proteins are found in heterocomplexes containing Hsp90, Hsp70͞Hsc70, the Hsp70 regulator Hip (p48), p60, various immunophilins, and the small acidic protein p23.A recent study proposed that the benzoquinone ansamycins geldanamycin (GA) and herbimycin A (HA), originally classified as tyrosine kinase inhibitors (12), do not execute their biological effects directly by inhibiting kinase activities, but rather indirectly by acting on Hsp90 (13). Thus, these agents provide a powerful tool to explore the physiological role of Hsp90. We show that Hsp90, in cooperation with Hsc70, p60, and other factors, mediates the refolding of t...