Structural-Thermodynamic Relationships of Interactions in the N-Terminal ATP-Binding Domain of Hsp90

Despite its importance as a target in anti-cancer therapeutics and the numerous rational-based inhibitor design efforts aimed at it, there are only limited data available on structural-thermodynamic relationships of interactions of the N-terminal ATP-binding domain of Hsp90 (N-Hsp90). Here, we redress this by presenting an investigation of binding of nucleotides and ansamycin compounds to this domain. Interactions of nucleotides with N-Hsp90 are relatively weak (> 10 μM) and are strongly enthalpy driven over the temperature range 10-25 °C. Geldanamycin (GA) and its analogues 17-AAG [17-(allylamino)-17-demethoxy-GA] and 17-DMAG (17-N,N-dimethylaminoethylamino-17-demethoxy-GA) bind more strongly and have a dominant favourable enthalpic contribution over the temperature range investigated. We investigated the temperature dependence of the enthalpic contribution to binding. We found that while the ansamycin compounds have the commonly observed negative value, the nucleotides show a negligible or even a positive ΔC_p of binding. These data represent the first observation of a single binding site for which interactions with different ligands result in both negative and positive ΔC_p values. By addressing the likely impact of the potential contributions from protein-ligand interactions, we are able to attribute the anomalous ΔC_p for the nucleotides largely to a change in the conformation of the domain structure and local motion in the lid region of N-Hsp90 with the concomitant exposure of hydrophobic amino acid side chains.     

A Novel C-Terminal Homologue of Aha1 Co-Chaperone Binds to Heat Shock Protein 90 and Stimulates Its ATPase Activity in Entamoeba histolytica

Cytosolic heat shock protein 90 (Hsp90) has been shown to be essential for many infectious pathogens and is considered a potential target for drug development. In this study, we have carried out biochemical characterization of Hsp90 from a poorly studied protozoan parasite of clinical importance, Entamoeba histolytica. We have shown that Entamoeba Hsp90 can bind to both ATP and its pharmacological inhibitor, 17-AAG (17-allylamino-17-demethoxygeldanamycin), with K_d values of 365.2 and 10.77 μM, respectively, and it has a weak ATPase activity with a catalytic efficiency of 4.12 × 10^− 4 min^− 1 μM^− 1. Using inhibitor 17-AAG, we have shown dependence of Entamoeba on Hsp90 for its growth and survival. Hsp90 function is regulated by various co-chaperones. Previous studies suggest a lack of several important co-chaperones in E. histolytica. In this study, we describe the presence of a novel homologue of co-chaperone Aha1 (activator of Hsp90 ATPase), EhAha1c, lacking a canonical Aha1 N-terminal domain. We also show that EhAha1c is capable of binding and stimulating ATPase activity of EhHsp90. In addition to highlighting the potential of Hsp90 inhibitors as drugs against amoebiasis, our study highlights the importance of E. histolytica in understanding the evolution of Hsp90 and its co-chaperone repertoire.     

Theileria-induced constitutive IKK activation is independent of functional Hsp90

The intracellular parasite Theileria induces uncontrolled proliferation and host cell transformation. Parasite-induced transformation is accompanied by constitutive activation of IkappaB kinase (IKK), resulting in permanently high levels of activated nuclear factor (NF)-kappaB. IKK activation pathways normally require heat shock protein 90 (Hsp90), a chaperone that regulates the stability and activity of signalling molecules and can be blocked by the benzoquinone ansamycin compound geldanamycin (GA). In Theileria-transformed cells, IkappaBalpha and p65 phosphorylation, NF-kappaB nuclear translocation and DNA binding activity are largely resistant to GA and also NF-kappaB-dependent reporter gene expression is only partly affected. Our findings indicate that parasite-induced IKK activity does not require functional Hsp90.     

Structural studies on the co-chaperone Hop and its complexes with Hsp90

The tetratricopeptide repeat domain (TPR)-containing co-chaperone Hsp-organising protein (Hop) plays a critical role in mediating interactions between Heat Shock Protein (Hsp)70 and Hsp90 as part of the cellular assembly machine. It also modulates the ATPase activity of both Hsp70 and Hsp90, thus facilitating client protein transfer between the two. Despite structural work on the individual domains of Hop, no structure for the full-length protein exists, nor is it clear exactly how Hop interacts with Hsp90, although it is known that its primary binding site is the C-terminal MEEVD motif. Here, we have undertaken a biophysical analysis of the structure and binding of Hop to Hsp90 using a variety of truncation mutants of both Hop and Hsp90, in addition to mutants of Hsp90 that are thought to modulate the conformation, in particular the N-terminal dimerisation of the chaperone. The results establish that whilst the primary binding site of Hop is the C-terminal MEEVD peptide of Hsp90, binding also occurs at additional sites in the C-terminal and middle domain. In contrast, we show that another TPR-containing co-chaperone, CyP40, binds solely to the C-terminus of Hsp90.Truncation mutants of Hop were generated and used to investigate the dimerisation interface of the protein. In good agreement with recently published data, we find that the TPR2a domain that contains the Hsp90-binding site is also the primary site for dimerisation. However, our results suggest that residues within the TPR2b may play a role. Together, these data along with shape reconstruction analysis from small-angle X-ray scattering measurements are used to generate a solution structure for full-length Hop, which we show has an overall butterfly-like quaternary structure.Studies on the nucleotide dependence of Hop binding to Hsp90 establish that Hop binds to the nucleotide-free, ‘open’ state of Hsp90. However, the Hsp90-Hop complex is weakened by the conformational changes that occur in Hsp90 upon ATP binding. Together, the data are used to propose a detailed model of how Hop may help present the client protein to Hsp90 by aligning the bound client on Hsp70 with the middle domain of Hsp90. It is likely that Hop binds to both monomers of Hsp90 in the form of a clamp, interacting with residues in the middle domain of Hsp90, thus preventing ATP hydrolysis, possibly by the prevention of association of N-terminal and middle domains in individual Hsp90 monomers.     

Heat Shock Protein 90 as a Drug Target against Protozoan Infections: BIOCHEMICAL CHARACTERIZATION OF HSP90 FROM PLASMODIUM FALCIPARUM AND TRYPANOSOMA EVANSI AND EVALUATION OF ITS INHIBITOR AS A CANDIDATE DRUG*

Using a pharmacological inhibitor of Hsp90 in cultured malarial parasite, we have previously implicated Plasmodium falciparum Hsp90 (PfHsp90) as a drug target against malaria. In this study, we have biochemically characterized PfHsp90 in terms of its ATPase activity and interaction with its inhibitor geldanamycin (GA) and evaluated its potential as a drug target in a preclinical mouse model of malaria. In addition, we have explored the potential of Hsp90 inhibitors as drugs for the treatment of Trypanosoma infection in animals. Our studies with full-length PfHsp90 showed it to have the highest ATPase activity of all known Hsp90s; its ATPase activity was 6 times higher than that of human Hsp90. Also, GA brought about more robust inhibition of PfHsp90 ATPase activity as compared with human Hsp90. Mass spectrometric analysis of PfHsp90 expressed in P. falciparum identified a site of acetylation that overlapped with Aha1 and p23 binding domain, suggesting its role in modulating Hsp90 multichaperone complex assembly. Indeed, treatment of P. falciparum cultures with a histone deacetylase inhibitor resulted in a partial dissociation of PfHsp90 complex. Furthermore, we found a well known, semisynthetic Hsp90 inhibitor, namely 17-(allylamino)-17-demethoxygeldanamycin, to be effective in attenuating parasite growth and prolonging survival in a mouse model of malaria. We also characterized GA binding to Hsp90 from another protozoan parasite, namely Trypanosoma evansi. We found 17-(allylamino)-17-demethoxygeldanamycin to potently inhibit T. evansi growth in a mouse model of trypanosomiasis. In all, our biochemical characterization, drug interaction, and animal studies supported Hsp90 as a drug target and its inhibitor as a potential drug against protozoan diseases.     

Genetic and Biochemical Analysis of p23 and Ansamycin Antibiotics in the Function of Hsp90-Dependent Signaling Proteins

The ubiquitous molecular chaperone Hsp90 acts in concert with a cohort of associated proteins to facilitate the functional maturation of a number of cellular signaling proteins, such as steroid hormone receptors and oncogene tyrosine kinases. The Hsp90-associated protein p23 is required for the assembly of functional steroid aporeceptor complexes in cell lysates, and Hsp90-binding ansamycin antibiotics disrupt the activity of Hsp90-dependent signaling proteins in cultured mammalian cells and prevent the association of p23 with Hsp90-receptor heterocomplexes; these observations have led to the hypotheses that p23 is required for the maturation of Hsp90 target proteins and that ansamycin antibiotics abrogate the activity of such proteins by disrupting the interaction of p23 with Hsp90. In this study, I demonstrate that ansamycin antibiotics disrupt the function of Hsp90 target proteins expressed in yeast cells; prevent the assembly of Sba1, a yeast p23-like protein, into steroid receptor-Hsp90 complexes; and result in the assembly of receptor-Hsp90 complexes that are defective for ligand binding. To assess the role of p23 in Hsp90 target protein function, I show that the activity of Hsp90 target proteins is unaffected by deletion of SBA1. Interestingly, steroid receptor activity in cells lacking Sba1 displays increased sensitivity to ansamycin antibiotics, and this phenotype is rescued by the expression of human p23 in yeast cells. These findings indicate that Hsp90-dependent signaling proteins can achieve a functional conformation in vivo in the absence of p23. Furthermore, while the presence of p23 decreases the sensitivity of Hsp90-dependent processes to ansamycin treatment, ansamycin antibiotics disrupt signaling through some mechanism other than altering the Hsp90-p23 interaction.     

Conformational dynamics of the molecular chaperone Hsp90 in complexes with a co-chaperone and anticancer drugs

The molecular chaperone Hsp90 is essential for the correct folding, maturation and activation of a diverse array of client proteins, including several key constituents of oncogenic processes. Hsp90 has become a focus of cancer research, since it represents a target for direct prophylaxis against multistep malignancy. Hydrogen-exchange mass spectrometry was used to study the structural and conformational changes undergone by full-length human Hsp90beta in solution upon binding of the kinase-specific co-chaperone Cdc37 and two Hsp90 ATPase inhibitors: Radicicol and the first-generation anticancer drug DMAG. Changes in hydrogen exchange pattern in the complexes in regions of Hsp90 remote to the ligand-binding site were observed indicating long-range effects. In particular, the interface between the N-terminal domain and middle domains exhibited significant differences between the apo and complexed forms. For the inhibitors, differences in the interface between the middle domain and the C-terminal domain were also observed. These data provide important insight into the structure of the biologically active form of the protein.     

The Non-canonical Hop Protein from Caenorhabditis elegans Exerts Essential Functions and Forms Binary Complexes with Either Hsc70 or Hsp90

Heat shock protein (Hsp) 70/Hsp90-organizing proteins (Hop/Sti1) are thought to function as adaptor proteins to link the two chaperone machineries Hsp70 and Hsp90 during the processing of substrate proteins in eukaryotes. Hop (Hsp70/Hsp90-organizing protein) is composed of three tetratricopeptide repeat (TPR) domains, of which the first (TPR1) binds to Hsp70, the second (TPR2A) binds to Hsp90, and the third (TPR2B) is of unknown function. Contrary to most other eukaryotes, the homologue closest to the Caenorhabditis elegans Hop homologue R09E12.3 (CeHop) lacks the TPR1 domain and the short linker region connecting it to TPR2A, questioning the reported function as an Hsp90/Hsp70 adaptor in vitro and in vivo. We observed high constitutive expression levels of CeHop and detected significant phenotypes upon knockdown, linking the protein to functions in gonad development. Interestingly, we observed physical interactions with both chaperones Hsp70 and Hsp90, albeit only the interaction with Hsp90 is strong and inhibition of the Hsp90 ATPase activity can be observed upon binding of CeHop. However, the formation of ternary complexes with both chaperone machineries is impaired, as Hsp70 and Hsp90 compete for CeHop interaction sites, in particular as Hsp90 binds to both TPR domains simultaneously and requires both TPR domains for ATPase regulation. These results imply that, at least in C. elegans, essential functions of Hop exist which apparently do not depend on the simultaneous binding of Hsp90 and Hsp70 to Hop.     

Hsp90, not Grp94, regulates the intracellular trafficking and stability of nascent ErbB2

The benzoquinone ansamycin geldanamycin (GA) stimulates proteasome-mediated degradation of plasma membrane-associated ErbB2, a receptor tyrosine kinase. Drug sensitivity is mediated by ErbB2's kinase domain and occurs subsequent to the disruption of Hsp90 interaction with this domain. Full-length ErbB2 is efficiently processed via the endoplasmic reticulum (ER) and Golgi network, so that at steady state most of the detectable protein is plasma membrane associated. However, previous studies have also demonstrated the GA sensitivity of newly synthesized ErbB2, normally a minor component of the total cellular pool of the kinase. Drug sensitivity of nascent ErbB2 is distinguished by 2 characteristics--protein instability and inability to traverse the ER. As nascent ErbB2 can associate with both cytoplasmic Hsp90 and its ER luminal homolog Grp 94, also a GA-binding protein, the purpose of this study was to examine the relative contributions of the cytoplasmic and ER luminal domains of ErbB2 to the GA sensitivity of the nascent kinase. By studying the drug sensitivity of ErbB2/DK, a construct lacking ErbB2's cytoplasmic kinase domain, and by examining the activity of a GA derivative that preferentially binds Hsp90, we conclude that both the stability and the maturation of nascent ErbB2 are regulated by its cytoplasmic, Hsp90-interacting domain.     

pH-Dependent Conformational Changes in Bacterial Hsp90 Reveal a Grp94-Like Conformation at pH 6 That Is Highly Active in Suppression of Citrate Synthase Aggregation

The molecular chaperone Hsp90 depends upon large conformational rearrangements for its function. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, structural and kinetic studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis shift the equilibrium between a preexisting set of conformational states in an organism-dependent manner. While many conformations of Hsp90 have been described, little is known about how they relate to chaperone function. In this study, we show that the conformational equilibrium of the bacterial Hsp90, HtpG, can be shifted with pH. Using small-angle X-ray scattering, we identify a two-state pH-dependent conformational equilibrium for apo HtpG. Our structural modeling reveals that this equilibrium is observed between the previously observed extended state and a second state that is strikingly similar to the recently solved Grp94 crystal structure. In the presence of nonhydrolyzable 5′-adenylyl-β,γ-imidodiphosphate, a third state, which is identical with the solved AMPPNP-bound structure from yeast Hsp90, is populated. Electron microscopy confirmed the observed conformational equilibria. We also identify key histidine residues that control this pH-dependent equilibrium; using mutagenesis, we successfully modulate the conformational equilibrium at neutral pH. Using these mutations, we show that the Grp94-like state provides stronger aggregation protection compared to the extended apo conformation in the context of a citrate synthase aggregation assay. These studies provide a more detailed view of HtpG's conformational dynamics and provide the first linkage between a specific conformation and chaperone function.     

Polypeptide release by Hsp90 involves ATP hydrolysis and is enhanced by the co-chaperone p23

The molecular chaperone Hsp90 binds and hydrolyses ATP, but how this ATPase activity regulates the interaction of Hsp90 with a polypeptide substrate is not yet understood. Using the glucocorticoid receptor ligand binding domain as a substrate, we show that dissociation of Hsp90 from bound polypeptide depends on the Hsp90 ATPase and is blocked by geldanamycin, a specific ATPase inhibitor. The co-chaperone p23 greatly stimulates Hsp90 substrate release with ATP, but not with the non-hydrolysable nucleotides ATPgammaS or AMP-PNP. Point mutants of Hsp90 with progressively lower ATPase rates are progressively slower in ATP-dependent substrate release but are still regulated by p23. In contrast, ATPase-inactive Hsp90 mutants release substrate poorly and show no p23 effect. These results outline an ATP-driven cycle of substrate binding and release for Hsp90 which differs from that of other ATP-driven chaperones. Conversion of the ATP state of Hsp90 to the ADP state through hydrolysis is required for efficient release of substrate polypeptide. p23 couples the ATPase activity to polypeptide dissociation and thus can function as a substrate release factor for Hsp90.     

Hsp90 phosphorylation,Wee1 and the cell cycle

Heat Shock Protein 90 (Hsp90) is an essential molecular chaperone in eukaryotic cells, and it maintains the functional conformation of a subset of proteins that are typically key components of multiple regulatory and signaling networks mediating cancer cell proliferation, survival, and metastasis. It is possible to selectively inhibit Hsp90 using natural products such as geldanamycin (GA) or radicicol (RD), which have served as prototypes for development of synthetic Hsp90 inhibitors. These compounds bind within the ADP/ATP-binding site of the Hsp90 N-terminal domain to inhibit its ATPase activity. As numerous N-terminal domain inhibitors are currently undergoing extensive clinical evaluation, it is important to understand the factors that may modulate in vivo susceptibility to these drugs. We recently reported that Wee1Swe1-mediated, cell cycle-dependent, tyrosine phosphorylation of Hsp90 affects GA binding and impacts cancer cell sensitivity to Hsp90 inhibition. This phosphorylation also affects Hsp90 ATPase activity and its ability to chaperone a selected group of clients, comprised primarily of protein kinases. Wee1 regulates the G2/M transition. Here we present additional data demonstrating that tyrosine phosphorylation of Hsp90 by Wee1Swe1 is important for Wee1Swe1 association with Hsp90 and for Wee1Swe1 stability. Yeast expressing non-phosphorylatable yHsp90-Y24F, like swe1? yeast, undergo premature nuclear division that is insensitive to G2/M checkpoint arrest. These findings demonstrate the importance of Hsp90 phosphorylation for proper cell cycle regulation.     

RIP1 mediates the protection of geldanamycin on neuronal injury induced by oxygen-glucose deprivation combined with zVAD in primary cortical neurons

Caspase-dependent apoptosis is considered one of the most important cell death pathways. When the apoptotic process is blocked, a form of programmed necrosis called necroptosis occurs. Apoptosis and necroptosis may share some regulatory mechanisms. Recent studies indicated that receptor interacting protein 1 (RIP1), an Hsp90-associated kinase, is an important regulatory switch between apoptosis and necroptosis. In this study, we showed that oxygen-glucose deprivation (OGD) combined with a caspase inhibitor zVAD (OGD/zVAD)-induced RIP1 protein expression in a time-dependent manner. We found that geldanamycin (GA), a benzoquinone ansamycin, protected against neuronal injury induced by OGD/zVAD treatment in cultured primary neurons. More importantly, GA decreased RIP1 protein level in a time- and concentration-dependent manner. In this study, we found that GA also decreased the Hsp90 protein level, which caused instability of RIP1 protein, resulting in decreased RIP1 protein level but not RIP1 mRNA level after GA treatment. We concluded that the GA-mediated protection against OGD/zVAD-induced neuronal injury was associated with enhanced RIP1 protein instability by decreasing Hsp90 protein level. GA and its derivatives may be promising for the prevention of neuronal injury during ischemic injury.     

Elucidating the Mechanism of Substrate Recognition by the Bacterial Hsp90 Molecular Chaperone

Hsp90 is a conformationally dynamic molecular chaperone known to promote the folding and activation of a broad array of protein substrates (“clients”). Hsp90 is believed to preferentially interact with partially folded substrates, and it has been hypothesized that the chaperone can significantly alter substrate structure as a mechanism to alter the substrate functional state. However, critically testing the mechanism of substrate recognition and remodeling by Hsp90 has been challenging. Using a partially folded protein as a model system, we find that the bacterial Hsp90 adapts its conformation to the substrate, forming a binding site that spans the middle and C-terminal domains of the chaperone. Cross-linking and NMR measurements indicate that Hsp90 binds to a large partially folded region of the substrate and significantly alters both its local and long-range structure. These findings implicate Hsp90's conformational dynamics in its ability to bind and remodel partially folded proteins. Moreover, native-state hydrogen exchange indicates that Hsp90 can also interact with partially folded states only transiently populated from within a thermodynamically stable, native-state ensemble. These results suggest a general mechanism by which Hsp90 can recognize and remodel native proteins by binding and remodeling partially folded states that are transiently sampled from within the native ensemble.     

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.     

Antibiotic radicicol binds to the N-terminal domain of Hsp90 and shares important biologic activities with geldanamycin

The molecular chaperone Hsp90 plays an essential role in the folding and function of important cellular proteins including steroid hormone receptors, protein kinases and proteins controlling the cell cycle and apoptosis. A 15 Å deep pocket region in the N-terminal domain of Hsp90 serves as an ATP/ADP-binding site and has also been shown to bind geldanamycin, the only specific inhibitor of Hsp90 function described to date. We now show that radicicol, a macrocyclic antifungal structurally unrelated to geldanamycin, also specifically binds to Hsp90. Moreover, radicicol competes with geldanamycin for binding to the N-terminal domain of the chaperone, expressed either by in vitro translation or as a purified protein, suggesting that radicicol shares the geldanamycin binding site. Radicicol, as does geldanamycin, also inhibits the binding of the accessory protein p23 to Hsp90, and interferes with assembly of the mature progesterone receptor complex. Radicicol does not deplete cells of Hsp90, but rather increases synthesis as well as the steady-state level of this protein, similar to a stress response. Finally, radicicol depletes SKBR3 cells of p 185^erbB2, Raf-1 and mutant p53, similar to geldanamycin. Radicicol thus represents a structurally unique antibiotic, and the first non-benzoquinone ansamycin, capable of binding to Hsp90 and interfering with its function.