Barry Panaretou

Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex

Hsp90 (heat shock protein of 90 kDa) is a ubiquitous molecular chaperone responsible for the assembly and regulation of many eukaryotic signalling systems and is 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 interactions between domains and between protein chains, the detailed conformational changes in the amino-terminal domain that accompany ATP binding, and the structural basis for stabilization 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 are coupled to the chaperone ATPase cycle.

ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo.

Hsp90 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.

Activation of the ATPase activity of Hsp90 by the stress-regulated cochaperone Aha1

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.

The ATPase cycle of Hsp90 drives a molecular ‘clamp’ via transient dimerization of the N-terminal domains

How 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‐PNP‐ promoted 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 temperature‐ sensitive (ts) mutant T101I had normal ATP affinity 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.

Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)- domain co-chaperones

The in vivo function of the heat shock protein 90 (Hsp90) molecular chaperone is dependent on the binding and hydrolysis of ATP, and on interactions with a variety of co‐chaperones containing tetratricopeptide repeat (TPR) domains. We have now analysed the interaction of the yeast TPR‐domain co‐chaperones Sti1 and Cpr6 with yeast Hsp90 by isothermal titration calorimetry, circular dichroism spectroscopy and analytical ultracentrifugation, and determined the effect of their binding on the inherent ATPase activity of Hsp90. Sti1 and Cpr6 both bind with sub‐micromolar affinity, with Sti1 binding accompanied by a large conformational change. Two co‐chaperone molecules bind per Hsp90 dimer, and Sti1 itself is found to be a dimer in free solution. The inherent ATPase activity of Hsp90 is completely inhibited by binding of Sti1, but is not affected by Cpr6, although Cpr6 can reactivate the ATPase activity by displacing Sti1 from Hsp90. Bound Sti1 makes direct contact with, and blocks access to the ATP‐binding site in the N‐terminal domain of Hsp90. These results reveal an important role for TPR‐domain co‐chaperones as regulators of the ATPase activity of Hsp90, showing that the ATP‐dependent step in Hsp90‐mediated protein folding occurs after the binding of the folding client protein, and suggesting that ATP hydrolysis triggers client‐protein release.

Structural and functional analysis of the middle segment of Hsp90: Implications for ATP hydrolysis and client protein and cochaperone interactions

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.

The mechanism of Hsp90 regulation by the protein kinase-specific cochaperone p50(cdc37).

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 Hsp90s 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.

Structural basis for recruitment of the ATPase activator Aha1 to the Hsp90 chaperone machinery

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 Hsp90 (273–530). 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.

Hsp90-Dependent Activation of Protein Kinases Is Regulated by Chaperone-Targeted Dephosphorylation of Cdc37

Summary Activation of protein kinase clients by the Hsp90 system is mediated by the cochaperone protein Cdc37. Cdc37 requires phosphorylation at Ser13, but little is known about the regulation of this essential posttranslational modification. We show that Ser13 of uncomplexed Cdc37 is phosphorylated in vivo, as well as in binary complex with a kinase (C-K), or in ternary complex with Hsp90 and kinase (H-C-K). Whereas pSer13-Cdc37 in the H-C-K complex is resistant to nonspecific phosphatases, it is efficiently dephosphorylated by the chaperone-targeted protein phosphatase 5 (PP5/Ppt1), which does not affect isolated Cdc37. We show that Cdc37 and PP5/Ppt1 associate in Hsp90 complexes in yeast and in human tumor cells, and that PP5/Ppt1 regulates phosphorylation of Ser13-Cdc37 in vivo, directly affecting activation of protein kinase clients by Hsp90-Cdc37. These data reveal a cyclic regulatory mechanism for Cdc37, in which its constitutive phosphorylation is reversed by targeted dephosphorylation in Hsp90 complexes.

Structural and functional coupling of Hsp90‐ and Sgt1‐centred multi‐protein complexes

Sgt1 is an adaptor protein implicated in a variety of processes, including formation of the kinetochore complex in yeast, and regulation of innate immunity systems in plants and animals. Sgt1 has been found to associate with SCF E3 ubiquitin ligases, the CBF3 kinetochore complex, plant R proteins and related animal Nod‐like receptors, and with the Hsp90 molecular chaperone. We have determined the crystal structure of the core Hsp90–Sgt1 complex, revealing a distinct site of interaction on the Hsp90 N‐terminal domain. Using the structure, we developed mutations in Sgt1 interfacial residues, which specifically abrogate interaction with Hsp90, and disrupt Sgt1‐dependent functions in vivo, in plants and yeast. We show that Sgt1 bridges the Hsp90 molecular chaperone system to the substrate‐specific arm of SCF ubiquitin ligase complexes, suggesting a role in SCF assembly and regulation, and providing multiple complementary routes for ubiquitination of Hsp90 client proteins.