Apo-Hsp90 coexists in two open conformational states in solution

Background information. Hsp90 (90 kDa heat‐shock protein) plays a key role in the folding and activation of many client proteins involved in signal transduction and cell cycle control. The cycle of Hsp90 has been intimately associated with large conformational rearrangements, which are nucleotide‐binding‐dependent. However, up to now, our understanding of Hsp90 conformational changes derives from structural information, which refers to the crystal states of either recombinant Hsp90 constructs or the prokaryotic homologue HtpG (Hsp90 prokaryotic homologue). Results and discussion. Here, we present the first nucleotide‐free structures of the entire eukaryotic Hsp90 (apo‐Hsp90) obtained by small‐angle X‐ray scattering and single‐particle cryo‐EM (cryo‐electron microscopy). We show that, in solution, apo‐Hsp90 is in a conformational equilibrium between two open states that have never been described previously. By comparing our cryo‐EM maps with HtpG and known Hsp90 structures, we establish that the structural changes involved in switching between the two Hsp90 apo‐forms require large movements of the NTD (N‐terminal domain) and MD (middle domain) around two flexible hinge regions. Conclusions. The present study shows, for the first time, the structure of the entire eukaryotic apo‐Hsp90, along with its intrinsic flexibility. Although large structural rearrangements, leading to partial closure of the Hsp90 dimer, were previously attributed to the binding of nucleotides, our results reveal that they are in fact mainly due to the intrinsic flexibility of Hsp90 dimer. Taking into account the preponderant role of the dynamic nature of the structure of Hsp90, we reconsider the Hsp90 ATPase cycle.     

Physical Interaction between Bacterial Heat Shock Protein (Hsp) 90 and Hsp70 Chaperones Mediates Their Cooperative Action to Refold Denatured Proteins

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.     

Constitutive Expression of Small Heat Shock Protein in an htpG Disruptant of the Cyanobacterium Synechococcus sp. PCC 7942

In cyanobacteria, a disruptant of hspA encoding a small heat shock protein homologue, shows decreased cell growth rates at moderately high temperatures, and loss of both basal and acquired thermo-tolerances, which resemble the phenotype of an htpG disruptant. In vitro studies have shown that both small heat shock protein and Hsp90 can bind and keep non-native proteins in a refolding-competent state under denaturing conditions. The aim of the present study is to elucidate whether constitutive expression of HspA can functionally replace HtpG, a prokaryotic homolog of Hsp90, in the cyanobacterium Synechococcus sp. PCC 7942. HspA did not improve the viability of the htpG disruptant at a lethal temperature, although it did that of the wild type. It did not improve an iron-starved phenotype of the mutant under normal growth conditions, a novel phenotype found in the present study. These results suggest that cellular function of HtpG may differ significantly from that of HspA.     

Ribosomal protein L2 associates with E. coli HtpG and activates its ATPase activity

Although eukaryotic Hsp90 has been studied extensively, the function of its bacterial homologue HtpG remains elusive. Here we report that 50S ribosomal protein L2 was found as an associated protein with His-tagged HtpG from Escherichia coli cultured in minimum medium at 45 °C. L2 specifically activated ATPase activity of HtpG, but other denatured proteins did not. The analysis using domain derivatives of HtpG and L2 showed that C-terminal domain of L2 and the middle to C-terminal domain of HtpG are important for interaction. At physiological salt concentration, L2 was denatured state and was recognized by HtpG as well as other chaperones, DnaK/DnaJ/GrpE and GroEL/GroES. The ATPase of HtpG at increasing concentration of L2 indicated that an L2 molecule bound to a dimer HtpG with apparent K_D of 0.3 μM at 100 mM KCl and 3.3 μM at 200 mM KCl.     

5′‐N‐ethylcarboxamidoadenosine is not a paralog‐specific Hsp90 inhibitor

The molecular chaperone Hsp90 facilitates the folding and modulates activation of diverse substrate proteins. Unlike other heat shock proteins such as Hsp60 and Hsp70, Hsp90 plays critical regulatory roles by maintaining active states of kinases, many of which are overactive in cancer cells. Four Hsp90 paralogs are expressed in eukaryotic cells: Hsp90α/β (in the cytosol), Grp94 (in the endoplasmic reticulum), Trap1 (in mitochondria). Although numerous Hsp90 inhibitors are being tested in cancer clinical trials, little is known about why different Hsp90 inhibitors show specificity among Hsp90 paralogs. The paralog specificity of Hsp90 inhibitors is likely fundamental to inhibitor efficacy and side effects. In hopes of gaining insight into this issue we examined NECA (5′‐N‐ethylcarboxamidoadenosine), which has been claimed to be an example of a highly specific ligand that binds to one paralog, Grp94, but not cytosolic Hsp90. To our surprise we find that NECA inhibits many different Hsp90 proteins (Grp94, Hsp90α, Trap1, yeast Hsp82, bacterial HtpG). NMR experiments demonstrate that NECA can bind to the N‐terminal domains of Grp94 and Hsp82. We use ATPase competition experiments to quantify the inhibitory power of NECA for different Hsp90 proteins. This scale: Hsp82 > Hsp90α > HtpG ≈ Grp94 > Trap1, ranks Grp94 as less sensitive to NECA inhibition. Because NECA is primarily used as an adenosine receptor agonist, our results also suggest that cell biological experiments utilizing NECA may have confounding effects from cytosolic Hsp90 inhibition.     

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.     

HtpG is essential for the thermal stress management in cyanobacteria.

The heat shock protein (Hsp) HtpG is a member of the Hsp90 protein family. We cloned a single-copy gene encoding a homologue of HtpG from the unicellular cyanobacterium Synechococcus sp. PCC 7942. Sequence alignment with HtpGs from other prokaryotes revealed unique features in the cyanobacterial HtpG primary sequence. A monocistronic mRNA of the htpG gene increased transiently in response to heat shock. In order to elucidate the role of HtpG in vivo, we inactivated the htpG gene by targeted mutagenesis. Although the mutation did not affect the photoautotrophic growth at 30 and 42 degrees C, the mutant cells were unable to grow at 45 degrees C. They lost both basal and acquired thermotolerances. These results indicate that HtpG plays an essential role for the thermal stress management in cyanobacteria, the first such an example for either a photosynthetic or a prokaryotic organism.     

Transformation by T-antigen and other oncogenes delays Hsp25 accumulation in heat shocked NIH 3T3 fibroblasts

We have recently reported that transformation of murine NIH 3T3 cells by v-fos oncogene interfered with Hsp70 and Hsp25 accumulation after heat shock. Here, we have investigated the effect mediated by other oncogenes on the accumulation of these stress proteins. We report that T-antigen transformation of NIH 3T3 cells delayed and reduced the accumulation of Hsp25 after heat shock and decreased the heat-mediated phosphorylation of this protein. This decreased level of Hsp25 correlated with a reduced accumulation of the corresponding mRNA and was related to T-antigen level. In contrast, T-antigen had no effect on the expression of the major stress protein Hsp70 nor did it interfere with the level of Hsp90 or Hsp60. We report also that v-src or Ha-ras oncogenes delayed Hsp25 accumulation after heat shock but that only v-src reduced the heat-induced phosphorylation of this protein. v-src, but not Ha-ras, interfered with Hsp70 expression and none of these oncogenes had an effect on Hsp60 or Hsp90 levels. Taken together, these observations suggest that an altered accumulation of Hsp25 after heat shock is a common characteristic of NIH 3T3 fibroblasts transformed by different oncogenes.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C resonance assignments for free and IEEVD peptide-bound forms of the tetratricopeptide repeat domain from the human E3 ubiquitin ligase CHIP

The ubiquitin ligase CHIP catalyzes covalent attachment of ubiquitin to unfolded proteins chaperoned by the heat shock proteins Hsp70/Hsc70 and Hsp90. CHIP interacts with Hsp70/Hsc70 and Hsp90 by binding of a C-terminal IEEVD motif found in Hsp70/Hsc70 and Hsp90 to the tetratricopeptide repeat (TPR) domain of CHIP. Although recruitment of heat shock proteins to CHIP via interaction with the CHIP-TPR domain is well established, alterations in structure and dynamics of CHIP upon binding are not well understood. In particular, the absence of a structure for CHIP-TPR in the free form presents a significant limitation upon studies seeking to rationally design inhibitors that may disrupt interactions between CHIP and heat shock proteins. Here we report the ¹H, ¹³C, and ¹⁵N backbone and side chain chemical shift assignments for CHIP-TPR in the free form, and backbone chemical shift assignments for CHIP-TPR in the IEEVD-bound form. The NMR resonance assignments will enable further studies examining the roles of dynamics and structure in regulating interactions between CHIP and the heat shock proteins Hsp70/Hsc70 and Hsp90.     

Characterisation of the <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> Hsp70–Hsp90 organising protein (PfHop)

Malaria is caused by Plasmodium species, whose transmission to vertebrate hosts is facilitated by mosquito vectors. The transition from the cold blooded mosquito vector to the host represents physiological stress to the parasite, and additionally malaria blood stage infection is characterised by intense fever periods. In recent years, it has become clear that heat shock proteins play an essential role during the parasite's life cycle. Plasmodium falciparum expresses two prominent heat shock proteins: heat shock protein 70 (PfHsp70) and heat shock protein 90 (PfHsp90). Both of these proteins have been implicated in the development and pathogenesis of malaria. In eukaryotes, Hsp70 and Hsp90 proteins are functionally linked by an essential adaptor protein known as the Hsp70–Hsp90 organising protein (Hop). In this study, recombinant P. falciparum Hop (PfHop) was heterologously produced in E. coli and purified by nickel affinity chromatography. Using specific anti-PfHop antisera, the expression and localisation of PfHop in P. falciparum was investigated. PfHop was shown to co-localise with PfHsp70 and PfHsp90 in parasites at the trophozoite stage. Gel filtration and co-immunoprecipitation experiments suggested that PfHop was present in a complex together with PfHsp70 and PfHsp90. The association of PfHop with both PfHsp70 and PfHsp90 suggests that this protein may mediate the functional interaction between the two chaperones.     

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.     

Differential interactions of p23 and the TPR-containing proteins Hop, Cyp40, FKBP52 and FKBP51 with Hsp90 mutants

Hsp90 is required for the normal function of steroid receptors, but its binding to steroid receptors is mediated by Hsc70 and several hsp-associated accessory proteins. An assortment of Hsp90 mutants were tested for their abilities to interact with each of the following accessories: Hop, Cyp40, FKBP52, FKBP51, and p23. Of the 11 Hsp90 mutants tested, all were defective to some extent in associating with progestin (PR) complexes. In every case, however, reduced PR binding correlated with a defect in binding of one or more accessories. Co-precipitation of mutant Hsp90 forms with individual accessories was used to map Hsp90 sequences required for accessory protein interactions. Mutation of Hsp90's highly conserved C-terminal EEVD to AAVD resulted in diminished interactions with several accessory proteins, most particularly with Hop. Deletion of amino acids 661–677 resulted in loss of Hsp90 dimerization and also caused diminished interactions with all accessory proteins. Binding of p23 mapped most strongly to the N-terminal ATP-binding domain of Hsp90 while binding of TPR proteins mapped to the C-terminal half of Hsp90. These results and others further suggest that the N- and C-terminal regions of Hsp90 maintain important conformational links through intramolecular interactions and/or intermolecular influences in homodimers.     

C. elegans STI-1, the Homolog of Sti1/Hop, Is Involved in Aging and Stress Response

Environmental and physiological stresses such as heat shock, oxidative stress, heavy metals, and pathogenic conditions induce cellular stress response. This response is often mediated by heat shock proteins that function as molecular chaperones. A stress-inducible cochaperone, Sti1/Hop (Hsp organizer protein), functions as an adaptor protein that simultaneously binds with Hsp70 and Hsp90 to transfer client proteins from Hsp70 to Hsp90. However, the biological role of STI-1 in vivo is poorly understood in metazoans. Here, we report the characterization of the Caenorhabditis elegans homolog of Sti1/Hop, which is approximately 56% identical with human STI-1. C. elegans STI-1 (CeSTI-1) is expressed in the pharynx, intestine, nervous system, and muscle from larvae to adults. Analysis of proteins immunoprecipitated with anti-STI-1 antibody by mass spectrometry revealed that CeSTI-1 can bind with both Hsp70 and Hsp90 homologs like its mammalian counterpart. sti-1 expression is elevated by heat stress, and an sti-1(jh125) null mutant shows decreased fertility under heat stress conditions. These mutants also show abnormally high lethality in extreme heat and may be functioning with DAF-16 in thermotolerance. In addition, sti-1(jh125) mutants have a shortened life span. Our results confirm that CeSTI-1 is a cochaperone protein that may maintain homeostatic functions during episodes of stress and can regulate longevity in nematodes.     

Hsp90·Cdc37 Complexes with Protein Kinases Form Cooperatively with Multiple Distinct Interaction Sites

Protein kinases are the most prominent group of heat shock protein 90 (Hsp90) clients and are recruited to the molecular chaperone by the kinase-specific cochaperone cell division cycle 37 (Cdc37). The interaction between Hsp90 and nematode Cdc37 is mediated by binding of the Hsp90 middle domain to an N-terminal region of Caenorhabditis elegans Cdc37 (CeCdc37). Here we map the binding site by NMR spectroscopy and define amino acids relevant for the interaction between CeCdc37 and the middle domain of Hsp90. Apart from these distinct Cdc37/Hsp90 interfaces, binding of the B-Raf protein kinase to the cochaperone is conserved between mammals and nematodes. In both cases, the C-terminal part of Cdc37 is relevant for kinase binding, whereas the N-terminal domain displaces the nucleotide from the kinase. This interaction leads to a cooperative formation of the ternary complex of Cdc37 and kinase with Hsp90. For the mitogen-activated protein kinase extracellular signal-regulated kinase 2 (Erk2), we observe that certain features of the interaction with Cdc37·Hsp90 are conserved, but the contribution of Cdc37 domains varies slightly, implying that different kinases may utilize distinct variations of this binding mode to interact with the Hsp90 chaperone machinery.     

Interaction of Hsp90AA1 with phospholipids stabilizes membranes under stress conditions

During heat shock conditions, structural changes in cellular membranes may lead to cell death. Hsp90AA1 and other heat shock proteins involved in membranes are responsible for protecting membrane stabilization. However, the membrane binding mechanism of Hsp90AA1 remains largely uncharacterized. In this study, we showed Hsp90AA1 interacts with phospholipid membrane with high affinity. Using the depth-dependent fluorescence-quenching with brominated lipids, we found Hsp90AA1 penetrated 10.7?Å into the hydrocarbon core of the lipid bilayer. Circular dichroism spectra studies showed Hsp90AA1 lost part of its α-helical structures upon interaction with phospholipid membrane. By assessing binding properties of the three Hsp90AA1 domains, we found Hsp90AA1 interacted into the lipid bilayer mainly toward its C-terminus domain (CTD). Using scanning electron microscopy, we examined the protection on host cell membrane by overexpressing Hsp90AA1. The results indicated Hsp90AA1 or Hsp90AA1-CTD expressing E. coli cells exhibited better membrane integrity compared to the control after thermal treatment. The following liposome leakage assay suggested the protection of Hsp90AA1 might due to its stabilization of the membrane lipid. Collectively, the present study demonstrates Hsp90AA1 embeds into the lipid bilayer through its C-terminal domain and the Hsp90AA1-lipid association potentially has a significant function in keeping membranes stabilization during stress conditions.     

Hsp90-containing multiprotein complexes in the eukaryotic microbe Achlya

In the oomycete fungus Achlya ambisexualis, hyphae of the male strain undergo sexual differentiation in the presence of the steroid hormone antheridiol. Earlier studies demonstrated that antheridiol binds with high affinity to a 9S multiprotein complex from A. ambisexualis cytosols. Although these complexes were found to contain the heat shock protein Hsp90, the other components were not known. It was of interest to determine if any of the other protein components in the Achlya Hsp90-heterocomplexes would be homologous to those found in the steroid receptor-Hsp90-heterocomplexes of vertebrates. Cytosolic proteins of 110 kDa, 74 kDa, 64 kDa, 61 kDa, 56 kDa, 47 kDa, 27 kDa and 23 kDa, were found in repeated trials, to co-immunoprecipitate with Achlya Hsp90. The 74 kDa protein was identified as the heat shock protein Hsp70, the 23 kDa protein was found to be related to the vertebrate protein p23 and the 56 kDa protein was found to be related to immunophilin FKBP51. All three of these proteins are components of the vertebrate receptor heterocomplexes. The 110 kDa, 61 kDa and 27 kDa proteins appeared to be unique to the Achlya complexes. Unlike the seven other proteins co-immunoprecipitating with Hsp90, the 61 kDa protein was observed only in the co-immunoprecipitates produced from in vitro translates of RNA isolated from antheridiol-treated mycelia.     

Design and biological testing of peptidic dimerization inhibitors of human Hsp90 that target the C-terminal domain

BackgroundSmall molecules targeting the dimerization interface of the C-terminal domain of Hsp90, a validated target for cancer treatment, have yet to be identified.MethodsThree peptides were designed with the aim to inhibit the dimerization of Hsp90. Computational and biophysical methods examined the α-helical structure for the three peptides. Based on the Autodisplay technology, a novel flow cytometer dimerization assay was developed to test inhibition of Hsp90 dimerization. Microscale thermophoresis was used to determine the K_D of the peptides towards the C-terminal domain of Hsp90.ResultsMD simulations and CD spectroscopy indicated an α-helical structure for two of the three peptides. By flow cytometer analysis, IC₅₀ values of 2.08 μM for peptide H2 and 8.96 μM for peptide H3 were determined. Dimer formation of the C-terminal dimerization domain was analyzed by microscale thermophoresis, and a K_D of 1.29 nM was determined. Furthermore, microscale thermophoresis studies demonstrated a high affinity binding of H2 and H3 to the C-terminal domain, with a K_D of 1.02 μM and 1.46 μM, respectively.ConclusionsThese results revealed the first peptidic inhibitors of Hsp90 dimerization targeting the C-terminal domain. Furthermore, it has been shown that these peptides bind to the C-terminal domain with a low micromolar affinity.General significanceThese results can be used to design and screen for small molecules that inhibit the dimerization of the C-terminal domain of Hsp90, which could open a new route for cancer therapy.