Cdc37-Hsp90 complexes are responsive to nucleotide-induced conformational changes and binding of further cofactors

Hsp90 is an ATP-dependent molecular chaperone, which facilitates the activation and stabilization of hundreds of client proteins in cooperation with a defined set of cofactors. Many client proteins are protein kinases, which are activated and stabilized by Hsp90 in cooperation with the kinase-specific co-chaperone Cdc37. Other Hsp90 co-chaperones, like the ATPase activator Aha1, also are implicated in kinase activation, and it is not yet clear how Cdc37 is integrated into Hsp90 co-chaperone complexes. Here, we studied the interaction between Cdc37, Hsp90, and other Hsp90 co-chaperones from the nematode Caenorhabditis elegans. Nematode Cdc37 binds with high affinity to Hsp90 and strongly inhibits the ATPase activity. In contrast to the human Hsp90 system, we observed binding of Cdc37 to open and closed Hsp90 conformations, potentially reflecting two different binding modes. Using a novel ultracentrifugation setup, which allows accurate analysis of multifactorial protein complexes, we show that cooperative and competitive interactions exist between other co-chaperones and Cdc37-Hsp90 complexes in the C. elegans system. We observed strong competitive interactions between Cdc37 and the co-chaperones p23 and Sti1, whereas the binding of the phosphatase Pph5 and the ATPase activator Aha1 to Cdc37-Hsp90 complexes is possible. The ternary Aha1-Cdc37-Hsp90 complex is disrupted by the nucleotide-induced closing reaction at the N terminus of Hsp90. This implies a carefully regulated exchange process of cofactors during the chaperoning of kinase clients by Hsp90.     

The 'active life' of Hsp90 complexes

Hsp90 forms a variety of complexes differing both in clientele and co-chaperones. Central to the role of co-chaperones in the formation of Hsp90 complexes is the delivery of client proteins and the regulation of the ATPase activity of Hsp90. Determining the mechanisms by which co-chaperones regulate Hsp90 is essential in understanding the assembly of these complexes and the activation and maturation of Hsp90's clientele. Mechanistically, co-chaperones alter the kinetics of the ATP-coupled conformational changes of Hsp90. The structural changes leading to the formation of a catalytically active unit involve all regions of the Hsp90 dimer. Their complexity has allowed different orthologues of Hsp90 to evolve kinetically in slightly different ways. The interaction of the cytosolic Hsp90 with a variety of co-chaperones lends itself to a complex set of different regulatory mechanisms that modulate Hsp90's conformation and ATPase activity. It also appears that the conformational switches of Hsp90 are not necessarily coupled under all circumstances. Here, I described different co-chaperone complexes and then discuss in detail the mechanisms and role that specific co-chaperones play in this. I will also discuss emerging evidence that post-translational modifications also affect the ATPase activity of Hsp90, and thus complex formation. Finally, I will present evidence showing how Hsp90's active site, although being highly conserved, can be altered to show resistance to drug binding, but still maintain ATP binding and ATPase activity. Such changes are therefore unlikely to significantly alter Hsp90's interactions with client proteins and co-chaperones. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

More than folding: localized functions of cytosolic chaperones

Compared with other chaperone systems, heat shock proteins Hsp70 and Hsp90 interact with a larger variety of co-chaperone proteins that regulate their activity or aid in the folding of specific substrate proteins. Although many co-chaperones are soluble cytosolic proteins, co-chaperone domains are also found in modular adaptor proteins, which are often localized to intracellular membranes or elements of the cytoskeleton. These specialized co-chaperones include auxilin, cysteine string protein, Tom70, UNC-45 and homologs of Bag-1. The localized co-chaperones can harness the ATP-dependent mechanisms of Hsp70 and Hsp90 to do conformational work in diverse functional contexts, including vesicle secretion and recycling, protein transport and the regulated assembly and/or disassembly of protein complexes. Such flexibility is unique to the cytosolic Hsp70 and Hsp90 chaperone system.     

Interaction of Heat Shock Protein 90 and the Co-chaperone Cpr6 with Ura2, a Bifunctional Enzyme Required for Pyrimidine Biosynthesis

The molecular chaperone heat shock protein 90 (Hsp90) is an essential protein required for the activity and stability of multiple proteins termed clients. Hsp90 cooperates with a set of co-chaperone proteins that modulate Hsp90 activity and/or target clients to Hsp90 for folding. Many of the Hsp90 co-chaperones, including Cpr6 and Cpr7, contain tetratricopeptide repeat (TPR) domains that bind a common acceptor site at the carboxyl terminus of Hsp90. We found that Cpr6 and Hsp90 interacted with Ura2, a protein critical for pyrimidine biosynthesis. Mutation or inhibition of Hsp90 resulted in decreased accumulation of Ura2, indicating it is an Hsp90 client. Cpr6 interacted with Ura2 in the absence of stable Cpr6-Hsp90 interaction, suggesting a direct interaction. However, loss of Cpr6 did not alter the Ura2-Hsp90 interaction or Ura2 accumulation. The TPR domain of Cpr6 was required for Ura2 interaction, but other TPR containing co-chaperones, including Cpr7, failed to interact with Ura2 or rescue CPR6-dependent growth defects. Further analysis suggests that the carboxyl-terminal 100 amino acids of Cpr6 and Cpr7 are critical for specifying their unique functions, providing new information about this important class of Hsp90 co-chaperones.     

Extracellular heat shock protein (Hsp)70 and Hsp90α assist in matrix metalloproteinase-2 activation and breast cancer cell migration and invasion

Breast cancer is second only to lung cancer in cancer-related deaths in women, and the majority of these deaths are caused by metastases. Obtaining a better understanding of migration and invasion, two early steps in metastasis, is critical for the development of treatments that inhibit breast cancer metastasis. In a functional proteomic screen for proteins required for invasion, extracellular heat shock protein 90 alpha (Hsp90α) was identified and shown to activate matrix metalloproteinase 2 (MMP-2). The mechanism of MMP-2 activation by Hsp90α is unknown. Intracellular Hsp90α commonly functions with a complex of co-chaperones, leading to our hypothesis that Hsp90α functions similarly outside of the cell. In this study, we show that a complex of co-chaperones outside of breast cancer cells assists Hsp90α mediated activation of MMP-2. We demonstrate that the co-chaperones Hsp70, Hop, Hsp40, and p23 are present outside of breast cancer cells and co-immunoprecipitate with Hsp90α in vitro and in breast cancer conditioned media. These co-chaperones also increase the association of Hsp90α and MMP-2 in vitro. This co-chaperone complex enhances Hsp90α-mediated activation of MMP-2 in vitro, while inhibition of Hsp70 in conditioned media reduces this activation and decreases cancer cell migration and invasion. Together, these findings support a model in which MMP-2 activation by an extracellular co-chaperone complex mediated by Hsp90α increases breast cancer cell migration and invasion. Our studies provide insight into a novel pathway for MMP-2 activation and suggest Hsp70 as an additional extracellular target for anti-metastatic drug development.     

Functions of the Hsp90 chaperone system: lifting client proteins to new heights

The molecular chaperone Hsp90 is an essential protein in eukaryotic organisms and is highly conserved throughout all kingdoms of life. It serves as a platform for the folding and maturation of many client proteins including protein kinases and steroid hormone receptors. To fulfill this task Hsp90 performs conformational changes driven by the hydrolysis of ATP. Further, it can resort to a broad set of co-chaperones, which fit the Hsp90 machinery to the needs of specific client proteins. During the last years the number of identified co-chaperones has been consistently rising, implying that the client spectrum of Hsp90 may be much more diverse and larger than currently known. Many cofactors contain a TPR-domain for interactions at the C-terminus of Hsp90 and in many cases their functions and client sets remain to be uncovered. Hsp90 is also a putative target to interfere with cancerous and infectious diseases. Thus the knowledge on more of its cellular functions would provide also more therapeutic options for the future. In this review we compile the current knowledge on the Hsp90 ATPase mechanism, cofactor regulation and prospects of Hsp90 inhibition.     

Hop: more than an Hsp70/Hsp90 adaptor protein

Molecular chaperones facilitate the correct folding of other proteins under physiological and stress conditions. Recently it has become evident that various co-chaperone proteins regulate the cellular functions of these chaperones, particularly Hsp70 and Hsp90. Hop is one of the most extensively studied co-chaperones that is able to directly associate with both Hsp70 and Hsp90. The current dogma proposes that Hop functions primarily as an adaptor that directs Hsp90 to Hsp70-client protein complexes in the cytoplasm. However, recent evidence suggests that Hop can also modulate the chaperone activities of these Hsps, and that it is not dedicated to Hsp70 and Hsp90. While the co-chaperone function of Hop within the cytoplasm has been extensively studied, its association with nuclear complexes and prion proteins remains to be elucidated. This article will review the structural features of Hop, and the evidence that its biological function is considerably broader than previously envisaged.     

Biochemical and structural studies of the interaction of Cdc37 with Hsp90

The heat shock protein Hsp90 plays a key, but poorly understood role in the folding, assembly and activation of a large number of signal transduction molecules, in particular kinases and steroid hormone receptors. In carrying out these functions Hsp90 hydrolyses ATP as it cycles between ADP- and ATP-bound forms, and this ATPase activity is regulated by the transient association with a variety of co-chaperones. Cdc37 is one such co-chaperone protein that also has a role in client protein recognition, in that it is required for Hsp90-dependent folding and activation of a particular group of protein kinases. These include the cyclin-dependent kinases (Cdk) 4/6 and Cdk9, Raf-1, Akt and many others. Here, the biochemical details of the interaction of human Hsp90 beta and Cdc37 have been characterised. Small angle X-ray scattering (SAXS) was then used to study the solution structure of Hsp90 and its complexes with Cdc37. The results suggest a model for the interaction of Cdc37 with Hsp90, whereby a Cdc37 dimer binds the two N-terminal domain/linker regions in an Hsp90 dimer, fixing them in a single conformation that is presumably suitable for client protein recognition.     

Regulation of Hsp90 ATPase activity by the co-chaperone Cdc37p/p50cdc37

In vivo activation of client proteins by Hsp90 depends on its ATPase-coupled conformational cycle and on interaction with a variety of co-chaperone proteins. For some client proteins the co-chaperone Sti1/Hop/p60 acts as a "scaffold," recruiting Hsp70 and the bound client to Hsp90 early in the cycle and suppressing ATP turnover by Hsp90 during the loading phase. Recruitment of protein kinase clients to the Hsp90 complex appears to involve a specialized co-chaperone, Cdc37p/p50(cdc37), whose binding to Hsp90 is mutually exclusive of Sti1/Hop/p60. We now show that Cdc37p/p50(cdc37), like Sti1/Hop/p60, also suppresses ATP turnover by Hsp90 supporting the idea that client protein loading to Hsp90 requires a "relaxed" ADP-bound conformation. Like Sti1/Hop/p60, Cdc37p/p50(cdc37) binds to Hsp90 as a dimer, and the suppressed ATPase activity of Hsp90 is restored when Cdc37p/p50(cdc37) is displaced by the immunophilin co-chaperone Cpr6/Cyp40. However, unlike Sti1/Hop/p60, which can displace geldanamycin upon binding to Hsp90, Cdc37p/p50(cdc37) forms a stable complex with geldanamycin-bound Hsp90 and may be sequestered in geldanamycin-inhibited Hsp90 complexes in vivo.     

Cns1 is an activator of the Ssa1 ATPase activity

Hsp90 is a key mediator in the folding process of a growing number of client proteins. The molecular chaperone cooperates with many co-chaperones and partner proteins to fulfill its task. In Saccharomyces cerevisiae, several co-chaperones of Hsp90 interact with Hsp90 via a tetratricopeptide repeat (TPR) domain. Here we show that one of these proteins, Cns1, binds both to Hsp90 and to the yeast Hsp70 protein Ssa1 with comparable affinities. This is reminiscent of Sti1, another TPR-containing co-chaperone. Unlike Sti1, Cns1 exhibits no influence on the ATPase of Hsp90. However, it activates the ATPase of Ssa1 up to 30-fold by accelerating the rate-limiting ATP hydrolysis step. This stimulating effect is mediated by the N-terminal TPR-containing part of Cns1, whereas the C-terminal part showed no effect. Competition experiments allow the conclusion that Hsp90 and Ssa1 compete for binding to the single TPR domain of Cns1. Taken together, Cns1 is a potent cochaperone of Ssa1. Our findings highlight the importance of the regulation of Hsp70 function in the context of the Hsp90 chaperone cycle.     

Sgt1p is a unique co-chaperone that acts as a client adaptor to link Hsp90 to Skp1p

Sgt1p is a conserved, essential protein required for kinetochore assembly in both yeast and animal cells. Sgt1p has homology to both TPR and p23 domains, sequences often found in proteins that interact with and regulate the molecular chaperone, Hsp90. The presence of these domains and the recent findings that Sgt1p interacts with Hsp90 has led to the speculation that Sgt1p and Hsp90 form a co-chaperone complex. To test this possibility, we have used purified recombinant proteins to characterize the in vitro interactions between yeast Sgt1p and Hsp82p (an Hsp90 homologue in yeast). We show that Sgt1p interacts directly with Hsp82p via its p23 homology region in a nucleotide-dependent manner. However, Sgt1p binding does not alter the enzymatic activity of Hsp82p, suggesting that it is distinct from other co-chaperones. We find that Sgt1p can form a ternary chaperone complex with Hsp82p and Sti1p, a well characterized Hsp90 co-chaperone. Sgt1p interacts with its binding partner Skp1p through its TPR domains and links Skp1p to the core Hsp82p-Sti1p co-chaperone complex. The multidomain nature of Sgt1p and its ability to bridge the interaction between Skp1p and Hsp82p argue that Sgt1p acts as a "client adaptor" recruiting specific clients to Hsp82p co-chaperone complexes.     

Functional interactions between Hsp90 and the co-chaperones Cns1 and Cpr7 in Saccharomyces cerevisiae

Hsp90 complexes contain a class of co-chaperones characterized by a tetratricopeptide repeat (TPR) domain, which mediates binding to a carboxyl-terminal EEVD region in Hsp90. Among Hsp90 TPR co-chaperones in Saccharomyces cerevisiae, only Cns1 is essential. The amino terminus of Cns1, which harbors the TPR domain, is sufficient for viability when overexpressed. In a screen for temperature-sensitive alleles of CNS1, we identified mutations resulting in substitutions of conserved residues in the TPR domain. Mutations in CNS1 disrupt in vitro and in vivo interaction with Hsp90 and reduce Hsp90 function, indicating that Cns1 is a bona fide co-chaperone. Genetic interactions between CNS1 and another Hsp90 co-chaperone, CPR7, suggest that the two co-chaperones share an essential role in the cell. Although both the TPR and the isomerase domains of the cyclophilin Cpr7 are required for viability of cns1 mutant cells, this requirement does not depend on the catalytic function of the isomerase domain. Instead, hydrophilic residues on the surface of this domain appear to be important for the common Cns1.Cpr7 function. Although both co-chaperones interact with Hsp90 primarily through the carboxyl terminus (EEVD), Cns1 and Cpr7 are mostly found in complexes distinct from Hsp90. EEVD is required for normal growth in cns1 mutant cells, demonstrating for the first time in vivo requirement for this conserved region of Hsp90. Overall, our findings reveal a considerable degree of complexity in the interactions not only between Hsp90 and its co-chaperones, but also among the co-chaperones themselves.     

The Co-Chaperone Hch1 Regulates Hsp90 Function Differently than Its Homologue Aha1 and Confers Sensitivity to Yeast to the Hsp90 Inhibitor NVP-AUY922

Hsp90 is a dimeric ATPase responsible for the activation or maturation of a specific set of substrate proteins termed ‘clients’. This molecular chaperone acts in the context of a structurally dynamic and highly regulated cycle involving ATP, co-chaperone proteins and clients. Co-chaperone proteins regulate conformational transitions that may be impaired in mutant forms of Hsp90. We report here that the in vivo impairment of commonly studied Hsp90 variants harbouring the G313S or A587T mutation are exacerbated by the co-chaperone Hch1p. Deletion of HCH1, but not AHA1, mitigates the temperature sensitive phenotype and high sensitivity to Hsp90 inhibitor drugs observed in Saccharomyces cerevisiae that express either of these two Hsp90 variants. Moreover, the deletion of HCH1 results in high resistance to Hsp90 inhibitors in yeast that express wildtype Hsp90. Conversely, the overexpression of Hch1p greatly increases sensitivity to Hsp90 inhibition in yeast expressing wildtype Hsp90. We conclude that despite the similarity between these two co-chaperones, Hch1p and Aha1p regulate Hsp90 function in distinct ways and likely independent of their roles as ATPase stimulators. We further conclude that Hch1p plays a critical role in regulating Hsp90 inhibitor drug sensitivity in yeast.     

Chaperoning checkpoint kinase 1 (Chk1), an Hsp90 client, with purified chaperones

Checkpoint kinase 1 (Chk1), a serine/threonine kinase that regulates DNA damage checkpoints, is destabilized when heat shock protein 90 (Hsp90) is inhibited, suggesting that Chk1 is an Hsp90 client. In the present work we examined the interplay between Chk1 and Hsp90 in intact cells, identified a source of unchaperoned Chk1, and report the in vitro chaperoning of Chk1 in reticulocyte lysates and with purified chaperones and co-chaperones. We find that bacterially expressed Chk1 is post-translationally chaperoned to an active kinase. This reaction minimally requires Hsp90, Hsp70, Hsp40, Cdc37, and the protein kinase CK2. The co-chaperone Hop, although not essential for the activation of Chk1 in vitro, enhanced the chaperoning process, whereas the co-chaperone p23 did not stimulate the chaperoning reaction. Additionally, we found that the C-terminal regulatory domain of Chk1 affects the association of Chk1 with Hsp90. Collectively these results provide new insights into Hsp90-dependent chaperoning of a client kinase and identify a novel, biochemically tractable model system that will be useful to further dissect the Hsp90-dependent chaperoning of this important and ubiquitous class of Hsp90 clients.     

The co-chaperone p23 arrests the Hsp90 ATPase cycle to trap client proteins

The action of the molecular chaperone Hsp90 is essential for the activation and assembly of an increasing number of client proteins. This function of Hsp90 has been proposed to be governed by conformational changes driven by ATP binding and hydrolysis. Association of co-chaperones and client proteins regulate the ATPase activity of Hsp90. Here, we have examined the inhibition of the ATPase activity of human Hsp90beta by one such co-chaperone, human p23. We demonstrate that human p23 interacts with Hsp90 in both the absence and presence of nucleotide with a higher affinity in the presence of the ATP analogue AMP-PNP. This is consistent with an analysis of the effect of p23 on the steady-state kinetics that revealed a mixed mechanism of inhibition. Mass spectrometry of the intact Hsp90.p23 complex determined the stoichiometry of binding to be one p23 to each subunit of the Hsp90 dimer. p23 was also shown to interact with a monomeric, truncated fragment of Hsp90, lacking the C-terminal homodimerisation domain, indicating dimerisation of Hsp90 is not a prerequisite for association with p23. Complex formation between Hsp90 and p23 increased the apparent affinity of Hsp90 for AMP-PNP and completely inhibited the ATPase activity. We propose a model where the role of p23 is to lock individual subunits of Hsp90 in an ATP-dependent conformational state that has a high affinity for client proteins.     

Protein quality control: U-box-containing E3 ubiquitin ligases join the fold

Molecular chaperones act with folding co-chaperones to suppress protein aggregation and refold stress damaged proteins. However, it is not clear how slowly folding or misfolded polypeptides are targeted for proteasomal degradation. Generally, selection of proteins for degradation is mediated by E3 ubiquitin ligases of the mechanistically distinct HECT and RING domain sub-types. Recent studies suggest that the U-box protein family represents a third class of E3 enzymes. CHIP, a U-box-containing protein, is a degradatory co-chaperone of heat-shock protein 70 (Hsp70) and Hsp90 that facilitates the polyubiquitination of chaperone substrates. These data indicate a model for protein quality control in which the interaction of Hsp70 and Hsp90 with co-chaperones that have either folding or degradatory activity helps to determine the fate of non-native cellular proteins.     

The Hsp90 chaperone machinery: conformational dynamics and regulation by co-chaperones

Hsp90 is a dimeric molecular chaperone required for the activation and stabilization of numerous client proteins many of which are involved in essential cellular processes like signal transduction pathways. This activation process is regulated by ATP-induced large conformational changes, co-chaperones and posttranslational modifications. For some co-chaperones, a detailed picture on their structures and functions exists, for others their contributions to the Hsp90 system is still unclear. Recent progress on the conformational dynamics of Hsp90 and how co-chaperones affect the Hsp90 chaperone cycle significantly increased our understanding of the gearings of this complex molecular machinery. This article is part of a Special Issue entitled: Heat Shock Protein 90 (Hsp90).     

Cofactor Tpr2 combines two TPR domains and a J domain to regulate the Hsp70/Hsp90 chaperone system

In the eukaryotic cytosol, Hsp70 and Hsp90 cooperate with various co-chaperone proteins in the folding of a growing set of substrates, including the glucocorticoid receptor (GR). Here, we analyse the function of the co-chaperone Tpr2, which contains two chaperone-binding TPR domains and a DnaJ homologous J domain. In vivo, an increase or decrease in Tpr2 expression reduces GR activation, suggesting that Tpr2 is required at a narrowly defined expression level. As shown in vitro, Tpr2 recognizes both Hsp70 and Hsp90 through its TPR domains, and its J domain stimulates ATP hydrolysis and polypeptide binding by Hsp70. Furthermore, unlike other co-chaperones, Tpr2 induces ATP-independent dissociation of Hsp90 but not of Hsp70 from chaperone-substrate complexes. Excess Tpr2 inhibits the Hsp90-dependent folding of GR in cell lysates. We propose a novel mechanism in which Tpr2 mediates the retrograde transfer of substrates from Hsp90 onto Hsp70. At normal levels substoichiometric to Hsp90 and Hsp70, this activity optimizes the function of the multichaperone machinery.