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...

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.

The endoplasmic reticulum (ER) is an important site for protein folding and maturation in eukaryotes. The cellular requirement to synthesize proteins within the ER is matched by its folding capacity. However, the physiological demands or aberrations in folding may result in an imbalance which can lead to the accumulation of misfolded protein, also known as “ER stress.” The unfolded protein response (UPR) is a cell-signaling system that readjusts ER folding capacity to restore protein homeostasis. The key UPR signal activator, IRE1, responds to stress by propagating the UPR signal from the ER to the cytosol. Here, we discuss the structural and molecular basis of IRE1 stress signaling, with particular focus on novel mechanistic advances. We draw a comparison between the recently proposed allosteric model for UPR induction and the role of Hsp70 during polypeptide import to the mitochondrial matrix.

Activation of many protein kinases depends on their interaction with the Hsp90 molecular chaperone system. Recruitment of protein kinase clients to the Hsp90 chaperone system is mediated by the cochaperone adaptor protein Cdc37, which acts as a scaffold, simultaneously binding protein kinases and Hsp90. We have now expressed and purified an Hsp90-Cdc37-Cdk4 complex, defined its stoichiometry, and determined its 3D structure by single-particle electron microscopy. Comparison with the crystal structure of Hsp90 allows us to identify the locations of Cdc37 and Cdk4 in the complex and suggests a mechanism by which conformational changes in the kinase are coupled to the Hsp90 ATPase cycle.

Structure of the Ire1 autophosphorylation complex and implications for the unfolded protein responseIn the endoplasmic reticulum, unfolded proteins stimulate Ire1 autophosphorylation and RNase activity. The crystal structure of the dephosphorylated kinase/RNase domain of human Ire1 bound to ADP provides insight into the autophosphorylation reaction.