Hsp90 C-terminal Domain Research Articles

FTIR Study Reveal Intrinsically Disordered Nature of Heat Shock Protein 90

Heat shock protein 90 (Hsp90) is a highly conserved chaperone protein that enables the proper folding of a large number of structurally diverse proteins (a.k.a., clients) in the crowded cytosolic environment and plays a key role in regulating the heat shock response. An outstanding question is how Hsp90 accommodates the structural diversity of a large cohort of client proteins? We report ATR FTIR study on structural properties of Hsp90 C-terminal domain (CTD) and their temperature dependences. Our data reveal that within a narrow temperature window, from 35o to 45 oC around physiological temperatures, Hsp90CTD exhibits significant increases in protein aggregation and increases in unordered structures. Binding of Hsp90 inhibitor Clorobiocin elevates the transition temperature of protein aggregation from 38 °C (a physiological temperature) to 65 °C (far above the physiological temperatures). In striking contrast, the Hsp90 N-terminal domain and middle domain (NTMD) demonstrates no protein aggregation from 20° to 90 °C. Despite the intrinsically disordered nature of Hsp90CTD, it retains a protected hydrophobic core at 40 °C. We introduce a hydrophobic interlock (HPI) model to account for the functional capability of intrinsically disordered Hsp90. Implications of these results and the HPI model will be discussed in the light of the structural dynamics and client diversity of Hsp90.

Abstract B89: Mechanistic assessment of heat shock network perturbation by novel chemotypes identified using chemogenomic screening

Abstract The 90-kDa heat shock protein 90 (Hsp90) is a chaperone protein responsible for the folding and activation of numerous proteins disregulated in various cancers that drive oncogenesis. Consequently, inhibition of Hsp90 is thought to be an attractive anti-cancer therapeutic strategy, though to date, clinical success has been limited. Hsp90 inhibition leads to simultaneous degradation of multiple oncogenic client proteins, giving “polypharmacologic” effects through the targeting of a single, highly networked protein. Hence, the development of small molecule inhibitors of Hsp90 may be effective anti-cancer agents that have reduced likelihood of development of resistance when compared to other less networked single protein targets. This study demonstrates identification and validation of a novel chemotype that perturbs the heat shock protein network. A liquid culture-based phenotypic screen employing Saccharomyces cerevisiae haploid deletions strains with known sensitivities to Hsp90 inhibitors by screening of the NCI diversity II chemical library set. The screen identified 9 hit compounds; 2 previously identified heat shock modulators and 7 novel heat shock modulator hits. To validate these novel compounds as heat shock network selective, we bioinformatically show that the NSC145366 compounds sensitivity profile in NCI tumor cell lines significantly correlates with other Hsp90 inhibitors. Also, biochemical analyses demonstrate NSC145366 has direct interactions with two cytoplasmic isoforms of human Hsp90 both in recombinant protein preparations and in crude tumor cell lysates. In addition, we show that the compound preferentially interacts with the Hsp90 C-terminal dimerization domain as opposed to many of the current N-terminal domain-based inhibitors. Further characterization of the compound interaction with Hsp90 and its effects on the heat shock cellular network are ongoing. This study demonstrates the utility of phenotypic screening to identify heat shock protein network modulators and the possibility inhibiting Hsp90 in a novel and different mode from previous inhibitors. Citation Format: Kourtney M. Goode, Fiona M. Thomas, Pete Pascuzzi, V. Jo Davisson, Tony R. Hazbun. Mechanistic assessment of heat shock network perturbation by novel chemotypes identified using chemogenomic screening. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B89.

Targeting the Hsp90 C-terminal domain by the chemically accessible dihydropyrimidinone scaffold.

Hsp90 C-terminal ligands are potential new anti-cancer drugs alternative to the more studied N-terminal inhibitors. Here we report the identification of a new dihydropyrimidinone binding the C-terminus, which is not structurally related to other well-known natural and nature-inspired inhibitors of this second druggable Hsp90 site.

The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop

Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90-binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous α-helical folds as determined by NMR. These, and especially DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A-TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide-binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C-terminal domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1-DP1 module may serve as an Hsp70-client delivery system for the TPR2A-TPR2B-DP2 segment, which is required for client activation in vivo.

Structural analysis of the interaction between Hsp90 and the tumor suppressor protein p53

In eukaryotes, the essential dimeric molecular chaperone Hsp90 is required for the activation and maturation of specific substrates such as steroid hormone receptors, tyrosine kinases and transcription factors. Hsp90 is involved in the establishment of cancer and has become an attractive target for drug design. Here we present a structural characterization of the complex between Hsp90 and the tumor suppressor p53, a key mediator of apoptosis whose structural integrity is crucial for cell-cycle control. Using biophysical methods, we show that the human p53 DNA-binding domain interacts with multiple domains of yeast Hsp90. p53 binds to the Hsp90 C-terminal domain in its native-like state in a charge-dependent manner, but it also associates weakly with binding sites in the middle and the N-terminal domains. The fine-tuned interplay between several Hsp90 domains provides the interactions required for efficient chaperoning of p53.

Stoichiometry and thermodynamics of the interaction between the C-terminus of human 90 kDa heat shock protein Hsp90 and the mitochondrial translocase of outer membrane Tom70

A large majority of the 1000–1500 proteins in the mitochondria are encoded by the nuclear genome, and therefore, they are translated in the cytosol in the form and contain signals to enable the import of proteins into the organelle. The TOM complex is the major translocase of the outer membrane responsible for preprotein translocation. It consists of a general import pore complex and two membrane import receptors, Tom20 and Tom70. Tom70 contains a characteristic TPR domain, which is a docking site for the Hsp70 and Hsp90 chaperones. These chaperones are involved in protecting cytosolic preproteins from aggregation and then in delivering them to the TOM complex. Although highly significant, many aspects of the interaction between Tom70 and Hsp90 are still uncertain. Thus, we used biophysical tools to study the interaction between the C-terminal domain of Hsp90 (C-Hsp90), which contains the EEVD motif that binds to TPR domains, and the cytosolic fragment of Tom70. The results indicate a stoichiometry of binding of one monomer of Tom70 per dimer of C-Hsp90 with a KD of 360±30nM, and the stoichiometry and thermodynamic parameters obtained suggested that Tom70 presents a different mechanism of interaction with Hsp90 when compared with other TPR proteins investigated.

Elucidation of the Hsp90 C-Terminal Inhibitor Binding Site

The Hsp90 chaperone machine is required for the folding, activation, and/or stabilization of more than 50 proteins directly related to malignant progression. Hsp90 contains small molecule binding sites at both its N- and C-terminal domains; however, limited structural and biochemical data regarding the C-terminal binding site is available. In this report, the small molecule binding site in the Hsp90 C-terminal domain was revealed by protease fingerprinting and photoaffinity labeling utilizing LC-MS/MS. The identified site was characterized by generation of a homology model for hHsp90α using the SAXS open structure of HtpG and docking the bioactive conformation of NB into the generated model. The resulting model for the bioactive conformation of NB bound to Hsp90α is presented herein.

ATP Binding to Hsp90 Is Sufficient for Effective Chaperoning of p53 Protein

Hsp90 is a ubiquitous, ATP-dependent chaperone, essential for eukaryotes. It possesses a broad spectrum of substrates, among which is the p53 transcription factor, encoded by a tumor-suppressor gene. Here, we elucidate the role of the adenine nucleotide in the Hsp90 chaperone cycle, by taking advantage of a unique in vitro assay measuring Hsp90-dependent p53 binding to the promoter sequence. E42A and D88N Hsp90β variants bind but do not hydrolyze ATP, whereas E42A has increased and D88N decreased ATP affinity, compared with WT Hsp90β. Nevertheless, both of these mutants interact with WT p53 with a similar affinity. Surprisingly, in the case of WT, but also E42A Hsp90β, the presence of ATP stimulates dissociation of Hsp90-p53 complexes and results in p53 binding to the promoter sequence. D88N Hsp90β is not efficient in both of these reactions. Using a trap version of the chaperonin GroEL, which irreversibly captures unfolded proteins, we show that Hsp90 chaperone action on WT p53 results in a partial unfolding of the substrate. The ATP-dependent dissociation of p53-Hsp90 complex allows further folding of p53 protein to an active conformation, able to bind to the promoter sequence. Furthermore, in support of these results, the overproduction of WT or E42A Hsp90β stimulates transcription from the WAF1 gene promoter in H1299 cells. Altogether, our research indicates that ATP binding to Hsp90β is a sufficient step for effective WT p53 client protein chaperoning.

Mechanistic Studies of Sansalvamide A-Amide: An Allosteric Modulator of Hsp90

Herein we show that San A-amide, a structurally unique molecule, influences a subset of cancer-related pathways involving Hsp90. We show that San A-amide specifically binds to the N-middle domain of Hsp90 allosterically disrupts the binding of proteins thought to interact with the Hsp90 C-terminal domain, while having no effect on an N-terminal domain client protein. This unique mechanism suggests that San A-amide is a potential tool for studying C-terminal binding proteins of Hsp90 as well as a promising lead in the development of new cancer therapeutics.

Characterization of Celastrol to Inhibit Hsp90 and Cdc37 Interaction

The molecular chaperone heat shock protein 90 (Hsp90) is required for the stabilization and conformational maturation of various oncogenic proteins in cancer. The loading of protein kinases to Hsp90 is actively mediated by the cochaperone Cdc37. The crucial role of the Hsp90-Cdc37 complex has made it an exciting target for cancer treatment. In this study, we characterize Hsp90 and Cdc37 interaction and drug disruption using a reconstituted protein system. The GST pull-down assay and ELISA assay show that Cdc37 binds to ADP-bound/nucleotide-free Hsp90 but not ATP-bound Hsp90. Celastrol disrupts Hsp90-Cdc37 complex formation, whereas the classical Hsp90 inhibitors (e.g. geldanamycin) have no effect. Celastrol inhibits Hsp90 ATPase activity without blocking ATP binding. Proteolytic fingerprinting indicates celastrol binds to Hsp90 C-terminal domain to protect it from trypsin digestion. These data suggest that celastrol may represent a new class of Hsp90 inhibitor by modifying Hsp90 C terminus to allosterically regulate its chaperone activity and disrupt Hsp90-Cdc37 complex.

(−)-Epigallocatechin-3-gallate Inhibits Hsp90 Function by Impairing Hsp90 Association with Cochaperones in Pancreatic Cancer Cell Line Mia Paca-2

(-)-Epigallocatechin-3-gallate [(-)-EGCG], the most abundant polyphenolic catechin in green tea, showed chemoprevention and anticancer activities. (-)-EGCG was reported to bind to the C-terminal domain of heat shock protein 90 (Hsp90). The purpose of this study is to investigate (-)-EGCG as a novel Hsp90 inhibitor to impair Hsp90 superchaperone complex for simultaneous downregulation of oncogenic proteins in pancreatic cancer cells. MTS assay showed that (-)-EGCG exhibited antiproliferative activity against pancreatic cancer cell line Mia Paca-2 in vitro with IC50 below 50 muM. (-)-EGCG increased caspase-3 activity up to 3-fold in a time- and concentration-dependent manner. Western blotting analysis demonstrated that (-)-EGCG induced downregulation of oncogenic Hsp90 client proteins by approximately 70-95%, including Akt, Cdk4, Raf-1, Her-2, and pERK. Co-immunoprecipitation showed that (-)-EGCG decreased the association of cochaperones p23 and Hsc70 with Hsp90 by more than 50%, while it had little effect on the ATP binding to Hsp90. Proteolytic fingerprinting assay confirmed direct binding between (-)-EGCG and the Hsp90 C-terminal domain. These data suggest that the binding of (-)-EGCG to Hsp90 impairs the association of Hsp90 with its cochaperones, thereby inducing degradation of Hsp90 client proteins, resulting antiproliferating effects in pancreatic cancer cells.

Novobiocin and additional inhibitors of the Hsp90 C-terminal nucleotide-binding pocket.

The 90 kDa heat shock proteins (Hsp90), which are integrally involved in cell signaling, proliferation, and survival, are ubiquitously expressed in cells. Many proteins in tumor cells are dependent upon the Hsp90 protein folding machinery for their stability, refolding, and maturation. Inhibition of Hsp90 uniquely targets client proteins associated with all six hallmarks of cancer. Thus, Hsp90 has emerged as a promising target for the treatment of cancer. Hsp90 exists as a homodimer, which contains three domains. The N-terminal domain contains an ATP-binding site that binds the natural products geldanamycin and radicicol. The middle domain is highly charged and has high affinity for co-chaperones and client proteins. Initial studies by Csermely and co-workers suggested a second ATP-binding site in the C-terminus of Hsp90. This C-terminal nucleotide binding pocket has been shown to not only bind ATP, but cisplatin, novobiocin, epilgallocatechin-3-gallate (EGCG) and taxol. The coumarin antibiotics novobiocin, clorobiocin, and coumermycin A1 were isolated from several streptomyces strains and exhibit potent activity against Gram-positive bacteria. These compounds bind type II topoisomerases, including DNA gyrase, and inhibit the enzyme-catalyzed hydrolysis of ATP. As a result, novobiocin analogues have garnered the attention of numerous researchers as an attractive agent for the treatment of bacterial infection. Novobiocin was reported to bind weakly to the newly discovered Hsp90 C-terminal ATP binding site ( approximately 700 M in SkBr3 cells) and induce degradation of Hsp90 client proteins. Structural modification of this compound has led to an increase of 1000-fold in activity in anti-proliferative assays. Recent studies of structure-activity relationship (SAR) by Renoir and co-workers highlighted the crucial role of the C-4 and/or C-7 positions of the coumarin and removal of the noviose moiety, which appeared to be essential for degradation of Hsp90 client proteins. Unlike the N-terminal ATP binding site, there is no reported co-crystal structure of Hsp90 C-terminus bound to any inhibitor. The Hsp90 C-terminal domain, however, is known to contain a conserved pentapeptide sequence (MEEVD) which is recognized by co-chaperones. Cisplatin is a platinum-containing chemotherapeutic used to treat various types of cancers, including testicular, ovarian, bladder, and small cell lung cancer. Most notably, cisplatin coordinates to DNA bases, resulting in cross-linked DNA, which prohibits rapidly dividing cells from duplicating DNA for mitosis. Itoh and co-workers reported that cisplatin decreases the chaperone activity of Hsp90. This group applied bovine brain cytosol to a cisplatin affinity column, eluted with cisplatin and detected Hsp90 in the eluent. Subsequent experiments indicated that cisplatin exhibits high affinity for Hsp90. Moreover Csermely and co-workers determined that the cisplatin binding site is located proximal to the C-terminal ATP binding site. EGCG is one of the active ingredients found in green tea. EGCG is known to inhibit the activity of many Hsp90-dependent client proteins, including telomerase, several kinases, and the aryl hydrocarbon receptor (AhR). Recently Gasiewicz and co-workers reported that EGCG manifests its antagonistic activity against AhR through binding Hsp90. Similar to novobiocin, EGCG was shown to bind the C-terminus of Hsp90. Unlike previously identified N-terminal Hsp90 inhibitors, EGCG does not appear to prevent Hsp90 from forming multiprotein complexes. Studies are currently underway to determine whether EGCG competes with novobiocin or cisplatin binding. Taxol, a well-known drug for the treatment of cancer, is responsible for the stabilization of microtubules and the inhibition of mitosis. Previous studies have shown that taxol induces the activation of kinases and transcription factors, and mimics the effect of bacterial lipopolysaccharide (LPS), an attribute unrelated to its tubulin-binding properties. Rosen and co-workers prepared a biotinylated taxol derivative and performed affinity chromatography experiments with lysates from both mouse brain and macrophage cell lines. These studies led to identification of two chaperones, Hsp70 and Hsp90, by mass spectrometry. In contrast to typical Hsp90-binding drugs, taxol exhibits a stimulatory response. Recently it was reported that the geldanamycin derivative 17-AAG behaves synergistically with taxol-induced apoptosis. This review describes the different C-terminal inhibitors of Hsp90, with specific emphasis on structure-activity relationship studies of novobiocin and their effects on anti-proliferative activity.

Dimerization of Hsp90 Is Required for in Vivo Function

Heat shock protein 90 (Hsp90) plays a central role in signal transduction and has emerged as a promising target for anti-cancer therapeutics, but its molecular mechanism is poorly understood. At physiological concentration, Hsp90 predominantly forms dimers, but the function of full-length monomers in cells is not clear. Hsp90 contains three domains: the N-terminal and middle domains contribute directly to ATP binding and hydrolysis and the C domain mediates dimerization. To study the function of Hsp90 monomers, we used a single-chain strategy that duplicated the C-terminal dimerization domain. This novel monomerization strategy had the dual effect of stabilizing the C domain to denaturation and hindering intermolecular association of the ATPase domain. The resulting construct was predominantly monomeric at physiological concentration and did not function to support yeast viability as the sole Hsp90. The monomeric construct was also defective at ATP hydrolysis and the activation of a kinase and steroid receptor substrate in yeast cells. The ability to support yeast growth was rescued by the addition of a coiled-coil dimerization domain, indicating that the parental single-chain construct is functionally defective because it is monomeric.

Axon viability and mitochondrial function are dependent on local protein synthesis in sympathetic neurons.

(1) Axons contain numerous mRNAs and a local protein synthetic system that can be regulated independently of the cell body. (2) In this study, cultured primary sympathetic neurons were employed, to assess the effect of local protein synthesis blockade on axon viability and mitochondrial function. (3) Inhibition of local protein synthesis reduced newly synthesized axonal proteins by 65% and resulted in axon retraction after 6 h. Acute inhibition of local protein synthesis also resulted in a significant decrease in the membrane potential of axonal mitochondria. Likewise, blockade of local protein transport into the mitochondria by transfection of the axons with Hsp90 C-terminal domain decreased the mitochondrial membrane potential by 65%. Moreover, inhibition of the local protein synthetic system also reduced the ability of mitochondria to restore axonal levels of ATP after KCl-induced depolarization. (4) Taken together, these results indicate that the local protein synthetic system plays an important role in mitochondrial function and the maintenance of the axon.

The Bcl-2 Regulator FKBP38-Calmodulin-Ca2+ Is Inhibited by Hsp90

FKBP38 is a negative effector of the anti-apoptotic Bcl-2 protein in neuroblastoma cells. The interaction with Bcl-2 and the enzyme activity of FKBP38 depend on prior binding of calmodulin-Ca(2+) (CaM-Ca(2+)) at high Ca(2+) concentrations. The FKBP38 protein structure contains three tetratricopeptide repeat (TPR) motifs corresponding to the Hsp90 interaction sites of other immunophilins. In this study we show that the TPR domain of FKBP38 interacts with the C-terminal domain of Hsp90, but only if the FKBP38-CaM-Ca(2+) complex is preformed. Hence, FKBP38 is the first example of a TPR-containing immunophilin that interacts cofactor-dependently with Hsp90. In the ternary Hsp90-FKBP38-CaM-Ca(2+) complex the active site of FKBP38 is blocked, thus preventing interactions with Bcl-2. The dual control of the active site cleft of FKBP38 by CaM-Ca(2+) and Hsp90 highlights the importance of the enzyme activity of the FKBP38-CaM-Ca(2+) complex in the regulation of programmed cell death.

Unliganded and hormone-bound glucocorticoid receptors interact with distinct hydrophobic sites in the Hsp90 C-terminal domain

Unlike most chaperones, heat-shock protein 90 (Hsp90) interacts with a select group of "client proteins" that regulate essential biological processes. Little is known about how Hsp90 recognizes and binds these proteins. The glucocorticoid receptor (GR) is a well characterized Hsp90 client protein, whose hormone binding, nuclear-cytoplasmic trafficking, and transcriptional activity are regulated by Hsp90. Here, we provide evidence that unliganded and hormone-bound GR interact with two distinct, solvent-exposed hydrophobic sites in the Hsp90 C-terminal domain that contain the sequences "MxxIM" (HM10) and "L/MxxIL" (HM9). Our results indicate that binding of Hsp90 HM10 to unliganded GR stabilizes the unliganded ligand-binding pocket of GR indirectly by promoting an intramolecular interaction between the C-terminal alpha-helix (H12) and a solvent-exposed hydrophobic groove in the GR ligand binding domain. In the presence of hormone, Hsp90 appears to bind the hydrophobic groove of GR directly by mimicking the interactions of GR with transcriptional coactivators. The identified interactions provide insights into the mechanisms that enable Hsp90 to regulate the activity of both unliganded and hormone-bound GR and to sharpen the cellular response to hormone.

Modulation of Chaperone Function and Cochaperone Interaction by Novobiocin in the C-terminal Domain of Hsp90: EVIDENCE THAT COUMARIN ANTIBIOTICS DISRUPT Hsp90 DIMERIZATION

The C-terminal domain of Hsp90 displays independent chaperone activity, mediates dimerization, and contains the MEEVD motif essential for interaction with tetratricopeptide repeat-containing immunophilin cochaperones assembled in mature steroid receptor complexes. An alpha-helical region, upstream of the MEEVD peptide, helps form the dimerization interface and includes a hydrophobic microdomain that contributes to the Hsp90 interaction with the immunophilin cochaperones and corresponds to the binding site for novobiocin, a coumarin-related Hsp90 inhibitor. Mutation of selected residues within the hydrophobic microdomain significantly impacted the chaperone function of a recombinant C-terminal Hsp90 fragment and novobiocin inhibited wild-type chaperone activity. Prior incubation of the Hsp90 fragment with novobiocin led to a direct blockade of immunophilin cochaperone binding. However, the drug had little influence on the pre-formed Hsp90-immunophilin complex, suggesting that bound cochaperones mask the novobiocin-binding site. We observed a differential effect of the drug on Hsp90-immunophilin interaction, suggesting that the immunophilins make distinct contacts within the C-terminal domain to specifically modulate Hsp90 function. Novobiocin also precluded the interaction of full-length Hsp90 with the p50(cdc37) cochaperone, which targets the N-terminal nucleotide-binding domain, and is prevalent in Hsp90 complexes with protein kinase substrates. Novobiocin therefore acts locally and allosterically to induce conformational changes within multiple regions of the Hsp90 protein. We provide evidence that coumermycin A1, a coumarin structurally related to novobiocin, interferes with dimerization of the Hsp90 C-terminal domain. Coumarin-based inhibitors then may antagonize Hsp90 function by inducing a conformation favoring separation of the C-terminal domains and release of substrate.

Novobiocin induces a distinct conformation of Hsp90 and alters Hsp90-cochaperone-client interactions.

Hsp90 functions to facilitate the folding of newly synthesized and denatured proteins. Hsp90 function is modulated through its interactions with cochaperones and the binding and hydrolysis of ATP. Recently, novobiocin has been shown to bind to a second nucleotide binding site located within the C-terminal domain of Hsp90. In this report, we have examined the effect of novobiocin on Hsp90 function in reticulocyte lysate. Novobiocin specifically inhibited the maturation of the heme-regulated eIF2alpha kinase (HRI) in a concentration-dependent manner. Novobiocin induced the dissociation of Hsp90 and Cdc37 from immature HRI, while the Hsp90 cochaperones p23, FKBP52, and protein phosphatase 5 remained associated with immature HRI. Proteolytic fingerprinting of Hsp90 indicated that novobiocin had a distinct effect on the conformation of Hsp90, and molybdate lowered the concentration of novobiocin required to alter Hsp90's conformation by 10-fold. The recombinant C-terminal domain of Hsp90 adopted a proteolytic resistant conformation in the presence of novobiocin, indicating that alteration of Hsp90/cochaperone interactions was not the cause of the novobiocin-induced protease resistance within Hsp90's C-terminal domain. The concentration dependence of this novobiocin-induced conformation change correlated with the dissociation of Hsp90 and Cdc37 from immature HRI and novobiocin-induced inhibition of Hsp90/Cdc37-dependent activation of HRI's autokinase activity. The data suggest that binding of novobiocin to the C-terminal nucleotide binding site of Hsp90 induces a change in Hsp90's conformation leading to the dissociation of bound kinase. The unique structure and properties of novobocin-bound Hsp90 suggest that it may represent the "client-release" conformation of the Hsp90 machine.

A hydrophobic segment within the C-terminal domain is essential for both client-binding and dimer formation of the HSP90-family molecular chaperone.

The alpha isoform of human 90-kDa heat shock protein (HSP90alpha) is composed of three domains: the N-terminal (residues 1-400); middle (residues 401-615) and C-terminal (residues 621-732). The middle domain is simultaneously associated with the N- and C-terminal domains, and the interaction with the latter mediates the dimeric configuration of HSP90. Besides one in the N-terminal domain, an additional client-binding site exists in the C-terminal domain of HSP90. The aim of the present study is to elucidate the regions within the C-terminal domain responsible for the bindings to the middle domain and to a client protein, and to define the relationship between the two functions. A bacterial two-hybrid system revealed that residues 650-697 of HSP90alpha were essential for the binding to the middle domain. An almost identical region (residues 657-720) was required for the suppression of heat-induced aggregation of citrate synthase, a model client protein. Replacement of either Leu665-Leu666 or Leu671-Leu672 to Ser-Ser within the hydrophobic segment (residues 662-678) of the C-terminal domain caused the loss of bindings to both the middle domain and the client protein. The interaction between the middle and C-terminal domains was also found in human 94-kDa glucose-regulated protein. Moreover, Escherichia coli HtpG, a bacterial HSP90 homologue, formed heterodimeric complexes with HSP90alpha and the 94-kDa glucose-regulated protein through their middle-C-terminal domains. Taken together, it is concluded that the identical region including the hydrophobic segment of the C-terminal domain is essential for both the client binding and dimer formation of the HSP90-family molecular chaperone and that the dimeric configuration appears to be similar in the HSP90-family proteins.

Binding of ATP to Heat Shock Protein 90: EVIDENCE FOR AN ATP-BINDING SITE IN THE C-TERMINAL DOMAIN

The presence of a nucleotide binding site on hsp90 was very controversial until x-ray structure of the hsp90 N-terminal domain, showing a nonconventional nucleotide binding site, appeared. A recent study suggested that the hsp90 C-terminal domain also binds ATP (Marcu, M. G., Chadli, A., Bouhouche, I., Catelli, M. G., and Neckers, L. M. (2000) J. Biol. Chem. 275, 37181-37186). In this paper, the interactions of ATP with native hsp90 and its recombinant N-terminal (positions 1-221) and C-terminal (positions 446-728) domains were studied by isothermal titration calorimetry, scanning differential calorimetry, and fluorescence spectroscopy. Results clearly demonstrate that hsp90 possesses a second ATP-binding site located on the C-terminal part of the protein. The association constant between this domain of hsp90 and ATP-Mg and a comparison with the binding constant on the full-length protein are reported for the first time. Secondary structure prediction revealed motifs compatible with a Rossmann fold in the C-terminal part of hsp90. It is proposed that this potential Rossmann fold may constitute the C-terminal ATP-binding site. This work also suggests allosteric interaction between N- and C-terminal domains of hsp90.