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...

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.

C.Prodromou and B.Panaretou contributed equally to this workHow the ATPase activity of Heat shock protein 90 (Hsp90) is coupled to client protein activation remains obscure. Using truncation and missense mutants of Hsp90, we analysed the structural implications of its ATPase cycle. C-terminal truncation mutants lacking inherent dimerization displayed reduced ATPase activity, but dimerized in the presence of 5¢-adenylamido-diphosphate (AMP-PNP), and AMP-PNPpromoted association of N-termini in intact Hsp90 dimers was demonstrated. Recruitment of p23/Sba1 to C-terminal truncation mutants also required AMP-PNP-dependent dimerization. The temperaturesensitive (ts) mutant T101I had normal ATP af®nity but reduced ATPase activity and AMP-PNP-dependent N-terminal association, whereas the ts mutant T22I displayed enhanced ATPase activity and AMP-PNP-dependent N-terminal dimerization, indicating a close correlation between these properties. The locations of these residues suggest that the conformation of the`lid' segment (residues 100±121) couples ATP binding to N-terminal association. Consistent with this, a mutation designed to favour`lid' closure (A107N) substantially enhanced ATPase activity and N-terminal dimerization. These data show that Hsp90 has a molecular`clamp' mechanism, similar to DNA gyrase and MutL, whose opening and closing by transient N-terminal dimerization are directly coupled to the ATPase cycle.

Activation of client proteins by the Hsp90 molecular chaperone is dependent on binding and hydrolysis of ATP, which drives a molecular clamp via transient dimerization of the N-terminal domains. The crystal structure of the middle segment of yeast Hsp90 reveals considerable evolutionary divergence from the equivalent regions of other GHKL protein family members such as MutL and GyrB, including an additional domain of new fold. Using the known structure of the N-terminal nucleotide binding domain, a model for the Hsp90 dimer has been constructed. From this structure, residues implicated in the ATPase-coupled conformational cycle and in interactions with client proteins and the activating cochaperone Aha1 have been identified, and their roles functionally characterized in vitro and in vivo.

Client protein activation by Hsp90 involves a plethora of cochaperones whose roles are poorly defined. A ubiquitous family of stress-regulated proteins have been identified (Aha1, activator of Hsp90 ATPase) that bind directly to Hsp90 and are required for the in vivo Hsp90-dependent activation of clients such as v-Src, implicating them as cochaperones of the Hsp90 system. In vitro, Aha1 and its shorter homolog, Hch1, stimulate the inherent ATPase activity of yeast and human Hsp90. The identification of these Hsp90 cochaperone activators adds to the complex roles of cochaperones in regulating the ATPase-coupled conformational changes of the Hsp90 chaperone cycle.

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Recruitment of protein kinase clients to the Hsp90 chaperone involves the cochaperone p50(cdc37) acting as a scaffold, binding protein kinases via its N-terminal domain and Hsp90 via its C-terminal region. p50(cdc37) also has a regulatory activity, arresting Hsp90's ATPase cycle during client-protein loading. We have localized the binding site for p50(cdc37) to the N-terminal nucleotide binding domain of Hsp90 and determined the crystal structure of the Hsp90-p50(cdc37) core complex. Dimeric p50(cdc37) binds to surfaces of the Hsp90 N-domain implicated in ATP-dependent N-terminal dimerization and association with the middle segment of the chaperone. This interaction fixes the lid segment in an open conformation, inserts an arginine side chain into the ATP binding pocket to disable catalysis, and prevents trans-activating interaction of the N domains.

Hsp90 is a molecular chaperone essential for the activation and assembly of many key eukaryotic signalling and regulatory proteins. Hsp90 is assisted and regulated by co-chaperones that participate in an ordered series of dynamic multiprotein complexes, linked to Hsp90s conformationally coupled ATPase cycle. The co-chaperones Aha1 and Hch1 bind to Hsp90 and stimulate its ATPase activity. Biochemical analysis shows that this activity is dependent on the N-terminal domain of Aha1, which interacts with the central segment of Hsp90. The structural basis for this interaction is revealed by the crystal structure of the N-terminal domain (1-153) of Aha1 (equivalent to the whole of Hch1) in complex with the middle segment of . Structural analysis and mutagenesis show that binding of N-Aha1 promotes a conformational switch in the middle-segment catalytic loop (370-390) of Hsp90 that releases the catalytic Arg 380 and enables its interaction with ATP in the N-terminal nucleotide-binding domain of the chaperone.