Hsp90 is a ubiquitous molecular chaperone responsible for assembly and regulation of many eukaryotic signalling systems, and an emerging target for rational chemotherapy of many cancers. Although the structures of isolated domains of Hsp90 have been determined, the arrangement and ATP-dependent dynamics of these in the full Hsp90 dimer have been elusive and contentious. Here we present the crystal structure of full-length yeast Hsp90 in complex with an ATP analogue and the co-chaperone p23/Sba1. The structure reveals the complex architecture of the 'closed' state of the Hsp90 chaperone, the extensive inter-domain and inter-strand interactions, the detailed conformational changes in the N-terminal domain that accompany ATP binding, and the structural basis for stabilisation of the closed state by p23/Sba1. Contrary to expectations, the closed Hsp90 would not enclose its client proteins but provides a bipartite binding surface whose formation and disruption is coupled to the chaperone ATPase cycle.Hsp90 is an essential molecular chaperone in eukaryotes, required for activation of many regulatory and signalling 'client' proteins. Hsp90 function depends on its ability to bind and hydrolyse ATP1, 2, and pharmacological inhibition by ATP-competitors promotes client degradation3, 4. The requirement of Hsp90 for the function of oncogenic protein kinases such as ErbB2, Cdk4, B-Raf, Akt/PKB etc (reviewed in5) makes it an attractive target for novel cancer therapeutics6. Hsp90 associates with a plethora of co-chaperones, several of which regulate progress through its ATPase cycle7-9. The p23 co-chaperone and its S.cerevisiae homologue Sba1, preferentially bind Hsp90 in the presence of ATP or ATP- Previous studies suggested that the dimeric Hsp90 operates a 'molecular clamp' mechanism coupled to its ATPase cycle, involving closure of a 'lid' segment and transient dimerisation of the N-terminal nucleotide-binding domain in the ATP-bound state17, 18. However, this model has recently been challenged19-21. We have now determined the crystal structure of yeast Hsp90 trapped in a closed conformation, in complex with a non-hydrolysable ATP analogue and p23/Sba1. The structure provides a first view of Hsp90 in the ATP-bound state, defining the conformational changes in the N-domain that accompany closure, and revealing how p23/Sba1 recognises and stabilises the ATP-bound conformation of the Hsp90 dimer. The structure confirms the ATPase coupled molecular clamp mechanism, and provides a structural basis from which to understand ATP-dependent activation of Hsp90 client proteins. Architecture of the Hsp90-p23/Sba1 ComplexYeast Hsp90, with an Ala107Asn mutation shown to activate Hsp90s ATPase cycle18, and with truncation of the dispensable charged-linker connecting the N-domain and middle segments22, was co-crystallised with the non-hydrolysable ATP analogue AMP-PNP and Sba1, the yeast homologue of p2311. Crystals were phased by molecular replacement with the isolated N-terminal domain23 and middle segment of yeast Hsp9024, and...

Heat shock protein 90 (Hsp90) is a molecular chaperone essential for activating many signaling proteins in the eukaryotic cell. Biochemical and structural analysis of Hsp90 has revealed a complex mechanism of ATPase-coupled conformational changes and interactions with cochaperone proteins, which facilitate activation of Hsp90's diverse "clientele." Despite recent progress, key aspects of the ATPase-coupled mechanism of Hsp90 remain controversial, and the nature of the changes, engendered by Hsp90 in client proteins, is largely unknown. Here, we discuss present knowledge of Hsp90 structure and function gleaned from crystallographic studies of individual domains and recent progress in obtaining a structure for the ATP-bound conformation of the intact dimeric chaperone. Additionally, we describe the roles of the plethora of cochaperones with which Hsp90 cooperates and growing insights into their biochemical mechanisms, which come from crystal structures of Hsp90 cochaperone complexes.

The cellular activity of several regulatory and signal transduction proteins, which depend on the Hsp90 molecular chaperone for folding, is markedly decreased by geldanamycin and by radicicol (monorden). We now show that these unrelated compounds both bind to the N-terminal ATP/ADP-binding domain of Hsp90, with radicicol displaying nanomolar affinity, and both inhibit the inherent ATPase activity of Hsp90 which is essential for its function in vivo. Crystal structure determinations of Hsp90 N-terminal domain complexes with geldanamycin and radicicol identify key aspects of their nucleotide mimicry and suggest a rational basis for the design of novel antichaperone drugs.

B.Panaretou and C.Prodromou contributed equally to this workHsp90 is an abundant molecular chaperone essential to the establishment of many cellular regulation and signal transduction systems, but remains one of the least well described chaperones. The biochemical mechanism of protein folding by Hsp90 is poorly understood, and the direct involvement of ATP has been particularly contentious. Here we demonstrate in vitro an inherent ATPase activity in both yeast Hsp90 and the Escherichia coli homologue HtpG, which is sensitive to inhibition by the Hsp90-specific antibiotic geldanamycin. Mutations of residues implicated in ATP binding and hydrolysis by structural studies abolish this ATPase activity in vitro and disrupt Hsp90 function in vivo. These results show that Hsp90 is directly ATP dependent in vivo, and suggest an ATP-coupled chaperone cycle for Hsp90-mediated protein folding.

CHIP is a dimeric U box E3 ubiquitin ligase that binds Hsp90 and/or Hsp70 via its TPR-domain, facilitating ubiquitylation of chaperone bound client proteins. We have determined the crystal structure of CHIP bound to an Hsp90 C-terminal decapeptide. The structure explains how CHIP associates with either chaperone type and reveals an unusual asymmetric homodimer in which the protomers adopt radically different conformations. Additionally, we identified CHIP as a functional partner of Ubc13-Uev1a in formation of Lys63-linked polyubiquitin chains, extending CHIP's roles into ubiquitin regulation as well as targeted destruction. The structure of Ubc13-Uev1a bound to the CHIP U box domain defines the basis for selective cooperation of CHIP with specific ubiquitin-conjugating enzymes. Remarkably, the asymmetric arrangement of the TPR domains in the CHIP dimer occludes one Ubc binding site, so that CHIP operates with half-of-sites activity, providing an elegant means for coupling a dimeric chaperone to a single ubiquitylation system.