The ER Chaperones BiP and Grp94 Regulate the Formation of Insulin-Like Growth Factor 2 (IGF2) Oligomers

While cytosolic Hsp90 chaperones have been extensively studied, less is known about how the ER Hsp90 paralog Grp94 recognizes clients and influences client folding. Here, we examine how Grp94 and the ER Hsp70 paralog, BiP, influence the folding of insulin-like growth factor 2 (IGF2), an established client protein of Grp94. ProIGF2 is composed of a disulfide-bonded insulin-like hormone and a C-terminal E-peptide that has sequence characteristics of an intrinsically disordered region. BiP and Grp94 have a minimal influence on folding whereby both chaperones slow proIGF2 folding and do not substantially alter the disulfide-bonded folding intermediates, suggesting that BiP and Grp94 may have an additional influence unrelated to proIGF2 folding. Indeed, we made the unexpected discovery that the E-peptide region allows proIGF2 to form dynamic oligomers. ProIGF2 oligomers can transition from a dynamic state that is capable of exchanging monomers to an irreversibly aggregated state, providing a plausible role for BiP and Grp94 in regulating proIGF2 oligomerization. In contrast to the modest influence on folding, BiP and Grp94 have a stronger influence on proIGF2 oligomerization and these chaperones exert counteracting effects. BiP suppresses proIGF2 oligomerization while Grp94 can enhance proIGF2 oligomerization in a nucleotide-dependent manner. We propose that BiP and Grp94 regulate the assembly and dynamic behavior of proIGF2 oligomers, although the biological role of proIGF2 oligomerization is not yet known.     

The hsp110 and Grp1 70 stress proteins: newly recognized relatives of the Hsp70s

Both the Grp170 and Hsp110 families represent relatively conserved and distinct sets of stress proteins, within a more diverse category that also includes the Hsp70s. All of these families are found in a wide variety of organisms from yeasts to humans. Although Hsp110s or Grp170s are not Hsp70s any more than Hsp70s are Hsp110s or Grp170s, it is still reasonable to refer to this combination of related families as the Hsp70 superfamily based on arguments discussed above and since no obvious prokaryotic Hsp110 or Grp170 has yet been identified. These proteins are related to their counterparts in the Hsp70/Grp78 family of eukaryotic stress proteins but are characterized by significantly larger molecular weights. The members of the Grp170 family are characterized by C-terminal ER retention sequences and are ER localized in yeasts and mammals. As a Grp, Grp170 is recognized to be coregulated with other major Grps by a well-known set of stress conditions, sometimes referred to as the unfolded protein response (Kozutsumi et al 1988; Nakaki et al 1989). The Hsp110 family members are localized in the nucleus and cytoplasm and, with other major Hsps, are also coregulated by a specific set of stress conditions, most notably including hyperthermic exposures. Hsp110 is sometimes called Hsp105, although it would be preferable to have a uniform term. The large Hsp70-like proteins are structurally similar to the Hsp70s but differ from them in important ways. In both the Grp170 and Hspl10 families, there is a long loop structure that is interposed between the peptide-binding ,-domain and the alpha-helical lid. In the Hsp110 family and Grp170, there are differing degrees of expansion in the alpha-helical domain and the addition of a C-terminal loop. This gives the appearance of much larger lid domains for Hsp110 and Grp170 compared with Hsp70. Both Hsp110 and Grp170 families have relatively conserved short sequences in the alpha-helical domain in the lid, which are conserved motifs in numerous proteins (we termed these motifs Magic and TedWylee as discussed earlier). The structural differences detailed in this review result in functional differences between the large (Grp170 and Hspl10) members of the Hsp70 superfamily, the most distinctive being an increased ability of these proteins to bind (hold) denatured polypeptides compared with Hsc70, perhaps related to the enlarged C-terminal helical domain. However, there is also a major difference between these large stress proteins; Hsp110 does not bind ATP in vitro, whereas Grp170 binds ATP avidly. The role of the Grp170 and Hsp110 stress proteins in cellular physiology is not well understood. Overexpression of Hsp110 in cultured mammalian cells increases thermal tolerance. Grp170 binds to secreted proteins in the ER and may be cooperatively involved in folding these proteins appropriately. These roles are similar to those of the Hsp70 family members, and, therefore, the question arises as to the differential roles played by the larger members of the superfamily. We have discussed evidence that the large members of the superfamily cooperate with members of the Hsp70 family, and these chaperones probably interact with a large number of chaperones and cochaperones in their functional activities. The fundamental point is that Hsp110 is found in conjunction with Hsp70 in the cytoplasm (and nucleus) and Grp170 is found in conjunction with78 in tha ER in every eucaryotic cell examined from yeast to humans. This would strongly argue that Hsp110 Grp170 exhibit functions in eucaryotes not effectively performed by Hsp70s or Grp78, respectively. Of interest in this respect is the observation that all Hsp110s loss of function or deletion mutants listed in the Drosophila deletion project database are lethal. The important task for the future is to determine the roles these conserved molecular chaperones play in normal and physiologically stressed cells.     

Interaction of radicicol with members of the heat shock protein 90 family of molecular chaperones.

The Hsp90 family of proteins in mammalian cells consists of Hsp90 alpha and beta, Grp94, and Trap-1 (Hsp75). Radicicol, an antifungal antibiotic that inhibits various signal transduction proteins such as v-src, ras, Raf-1, and mos, was found to bind to Hsp90, thus making it the prototype of a second class of Hsp90 inhibitors, distinct from the chemically unrelated benzoquinone ansamycins. We have used two novel methods to immobilize radicicol, allowing for detailed analyses of drug-protein interactions. Using these two approaches, we have studied binding of the drug to N-terminal Hsp90 point mutants expressed by in vitro translation. The results point to important drug contacts with amino acids inside the N-terminal ATP/ADP-binding pocket region and show subtle differences when compared with geldanamycin binding. Radicicol binds more strongly to Hsp90 than to Grp94, the Hsp90 homolog that resides in the endoplasmic reticulum. In contrast to Hsp90, binding of radicicol to Grp94 requires both the N-terminal ATP/ADP-binding domain as well as the adjacent negatively charged region. Radicicol also specifically binds to yeast Hsp90, Escherichia coli HtpG, and a newly described tumor necrosis factor receptor-interacting protein, Trap-1, with greater homology to bacterial HtpG than to Hsp90. Thus, the radicicol-binding site appears to be specific to and is conserved in all members of the Hsp90 family of molecular chaperones from bacteria to mammals, but is not present in other molecular chaperones with nucleotide-binding domains.     

The ATPase cycle of the endoplasmic chaperone Grp94

Grp94, the Hsp90 paralog of the endoplasmic reticulum, plays a crucial role in protein secretion. Like cytoplasmic Hsp90, Grp94 is regulated by nucleotide binding to its N-terminal domain. However, the question of whether Grp94 hydrolyzes ATP was controversial. This sets Grp94 apart from other members of the Hsp90 family where a slow but specific turnover of ATP has been unambiguously established. In this study we aimed at analyzing the nucleotide binding properties and the potential ATPase activity of Grp94. We show here that Grp94 has an ATPase activity comparable with that of yeast Hsp90 with a k(cat) of 0.36 min(-1) at 25 degrees C. Kinetic and equilibrium constants of the partial reactions of the ATPase cycle were determined using transient kinetic methods. Nucleotide binding appears to be tighter compared with other Hsp90s investigated, with dissociation constants (K(D)) of approximately 4 microm for ADP, ATP, and AMP-PCP. Interestingly, all nucleotides and inhibitors (radicicol, 5'-N-ethylcarboxamidoadenosine) studied here bind with similar rate constants for association (0.2-0.3 x 10(6) M(-1) s(-1)). Furthermore, there is a marked difference from cytosolic Hsp90s in that after binding, the ATP molecule does not seem to become trapped by conformational changes in Grp94. Grp94 stays predominantly in the open state concerning the nucleotide-binding pocket as evidenced by kinetic analyses. Thus, Grp94 shows mechanistically important differences in the interaction with adenosine nucleotides, but the basic hydrolysis reaction seems to be conserved between cytosolic and endoplasmic members of the Hsp90 family.     

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.     

The overexpression of Hsp90B1 is associated with tumorigenesis of canine mammary glands

Heat shock proteins (Hsp) are molecular chaperones that are responsible for protein folding and maintenance of cellular homeostasis. Hsp90, an important member of HSP family, has an important role in breast cancer. Glucose-regulated protein 94 (Grp94) is the endoplasmic reticulum paralog of Hsp90 encoded by Hsp90B1 gene. To test if this protein is overexpressed in dogs with mammary tumor, we estimated and compared its serum levels in healthy dogs and that of dogs with mammary tumors. Hsp90B1 mRNA expression was measured in tumorous and healthy mammary tissues (from age- and breed-matched dogs) by real-time PCR. The gene was found to be overexpressed in mammary tumors (3.586 ± 0.067 times). Further, it was heterologously expressed in a prokaryotic system as 90 kDa protein. A recombinant Grp94-based sandwich ELISA was developed to quantify serum Grp94 in dogs with mammary tumors. Based on receiver-operating characteristics’ analysis, the assay was found to be 90.62% sensitive and 93.75% specific for a cutoff value of 0.35 with respect to histopathological staining in diagnosing the disease. The t test showed that serum Grp94 levels were significantly elevated (92.97 ± 3.62 ng/ml) in dogs with mammary tumors compared with healthy controls (10.30 ± 0.79 ng/ml) (p < 0.0001). These findings suggest that Grp94 might act as a potential biomarker for prognosis of canine mammary tumors and monitoring its therapy.     

Grp94 Works Upstream of BiP in Protein Remodeling Under Heat Stress

Hsp90 and Hsp70 are highly conserved molecular chaperones that promote the proper folding and activation of substrate proteins that are often referred to as clients. The two chaperones functionally collaborate to fold specific clients in an ATP-dependent manner. In eukaryotic cytosol, initial client folding is done by Hsp70 and its co-chaperones, followed by a direct transfer of client refolding intermediates to Hsp90 for final client processing. However, the mechanistic details of collaboration of organelle specific Hsp70 and Hsp90 are lacking. This work investigates the collaboration of the endoplasmic reticulum (ER) Hsp70 and Hsp90, BiP and Grp94 respectively, in protein remodeling using in vitro refolding assays. We show that under milder denaturation conditions, BiP collaborates with its co-chaperones to refold misfolded proteins in an ATP-dependent manner. Grp94 does not play a major role in this refolding reaction. However, under stronger denaturation conditions that favor aggregation, Grp94 works in an ATP-independent manner to bind and hold misfolded clients in a folding competent state for subsequent remodeling by the BiP system. We also show that the collaboration of Grp94 and BiP is not simply a reversal of the eukaryotic refolding mechanism since a direct interaction of Grp94 and BiP is not required for client transfer. Instead, ATP binding but not hydrolysis by Grp94 facilitates the release of the bound client, which is then picked up by the BiP system for subsequent refolding in a Grp94-independent manner.     

A comprehensive study on the immunological reactivity of the Hsp90 molecular chaperone

Periodontitis is a chronic infectious disease, Porphyromonas gingivalis being the most implicated pathogen. In the present study, we investigated the role of P. gingivalis HtpG (PgHtpG), a bacterial ortholog of mammalian Hsp90, in the growth of P. gingivalis and also assessed the immunological cross-reactivity of the members of the Hsp90 family. Antiserum against rat liver Hsp90 potently reacted with yeast Hsp90, called Hsc82, and also weakly with human Hsp90 (hHsp90) and human mitochondrial paralog Trap1, but did not react with PgHtpG, Escherichia coli HtpG, or human endoplasmic reticulum paralog Grp94. Moreover, among 19 monoclonal antibodies raised against hHsp90, nine cross-reacted with yeast Hsc82, and one with human Grp94, but none bound to PgHtpG or E. coli HtpG. Among them, three mAbs that strongly reacted with yeast Hsc82 recognized Asn(291)-Ile(304), a conserved region of the family protein. The polyclonal antibody raised against a peptide, Met(315)-Glu(328), of human Grp94, which corresponded to the conserved region of hHsp90, cross-reacted with hHsp90, but not with other Hsp90-family members. Thus, although mammalian Hsp90 shares some immunological reactivity with yeast Hsc82, human Grp94, and human Trap1, it is considerably distinct from its bacterial ortholog, HtpG. Disruption of the P. gingivalis htpG gene neither affected bacterial survival nor altered the sensitivity of P. gingivalis to various forms of stress.     

Corresponding functional dynamics across the Hsp90 Chaperone family: insights from a multiscale analysis of MD simulations

Understanding how local protein modifications, such as binding small-molecule ligands, can trigger and regulate large-scale motions of large protein domains is a major open issue in molecular biology. We address various aspects of this problem by analyzing and comparing atomistic simulations of Hsp90 family representatives for which crystal structures of the full length protein are available: mammalian Grp94, yeast Hsp90 and E.coli HtpG. These chaperones are studied in complex with the natural ligands ATP, ADP and in the Apo state. Common key aspects of their functional dynamics are elucidated with a novel multi-scale comparison of their internal dynamics. Starting from the atomic resolution investigation of internal fluctuations and geometric strain patterns, a novel analysis of domain dynamics is developed. The results reveal that the ligand-dependent structural modulations mostly consist of relative rigid-like movements of a limited number of quasi-rigid domains, shared by the three proteins. Two common primary hinges for such movements are identified. The first hinge, whose functional role has been demonstrated by several experimental approaches, is located at the boundary between the N-terminal and Middle-domains. The second hinge is located at the end of a three-helix bundle in the Middle-domain and unfolds/unpacks going from the ATP- to the ADP-state. This latter site could represent a promising novel druggable allosteric site common to all chaperones.     

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.     

Structure of the N-terminal domain of GRP94. Basis for ligand specificity and regulation

GRP94, the endoplasmic reticulum (ER) paralog of the chaperone Hsp90, plays an essential role in the structural maturation or secretion of a subset of proteins destined for transport to the cell surface, such as the Toll-like receptors 2 and 4, and IgG, respectively. GRP94 differs from cytoplasmic Hsp90 by exhibiting very weak ATP binding and hydrolysis activity. GRP94 also binds selectively to a series of substituted adenosine analogs. The high resolution crystal structures at 1.75-2.1 A of the N-terminal and adjacent charged domains of GRP94 in complex with N-ethylcarboxamidoadenosine, radicicol, and 2-chlorodideoxyadenosine reveals a structural mechanism for ligand discrimination among hsp90 family members. The structures also identify a putative subdomain that may act as a ligand-responsive switch. The residues of the charged region fold into a disordered loop whose termini are ordered and continue the twisted beta sheet that forms the structural core of the N-domain. This continuation of the beta sheet past the charged domain suggests a structural basis for the association of the N-terminal and middle domains of the full-length chaperone.     

Characterization of the Grp94/OS-9 Chaperone–Lectin Complex

Grp94 is a macromolecular chaperone belonging to the hsp90 family and is the most abundant glycoprotein in the endoplasmic reticulum (ER) of mammals. In addition to its essential role in protein folding, Grp94 was proposed to participate in the ER-associated degradation quality control pathway by interacting with the lectin OS-9, a sensor for terminally misfolded proteins. To understand how OS-9 interacts with ER chaperone proteins, we mapped its interaction with Grp94. Glycosylation of the full-length Grp94 protein was essential for OS-9 binding, although deletion of the Grp94 N-terminal domain relieved this requirement suggesting that the effect was allosteric rather than direct. Although yeast OS-9 is composed of a well-established N-terminal mannose recognition homology lectin domain and a C-terminal dimerization domain, we find that the C-terminal domain of OS-9 in higher eukaryotes contains “mammalian-specific insets” that are specifically recognized by the middle and C-terminal domains of Grp94. Additionally, the Grp94 binding domain in OS-9 was found to be intrinsically disordered. The biochemical analysis of the interacting regions provides insight into the manner by which the two associate and it additionally hints at a plausible biological role for the Grp94/OS-9 complex.     

Sensitivity of mature Erbb2 to geldanamycin is conferred by its kinase domain and is mediated by the chaperone protein Hsp90

ErbB receptors are a family of ligand-activated tyrosine kinases that play a central role in proliferation, differentiation, and oncogenesis. ErbB2 is overexpressed in >25% of breast and ovarian cancers and is correlated with poor prognosis. Although ErbB2 and ErbB1 are highly homologous, they respond quite differently to geldanamycin (GA), an antibiotic that is a specific inhibitor of the chaperone protein Hsp90. Thus, although both mature and nascent ErbB2 proteins are down-regulated by GA, only nascent ErbB1 is sensitive to the drug. To reveal the underlying mechanism behind these divergent responses, we made a chimeric receptor (ErbB1/2) composed of the extracellular and transmembrane domains of ErbB1 and the intracellular domain of ErbB2. The ErbB1/2 protein is functional since its kinase activity was stimulated by epidermal growth factor. The sensitivity of ErbB1/2 to GA was similar to that of ErbB2 and unlike that of ErbB1, indicating that the intracellular domain of the chimera confers GA sensitivity. This finding also suggests that the GA sensitivity of mature ErbB2 depends on cytosolic Hsp90, rather than Grp94, a homolog of Hsp90 that is restricted to the lumen of the endoplasmic reticulum, although both chaperones bind to and are inhibited by GA. Lack of Grp94 involvement in mediating ErbB2 sensitivity to GA is further suggested by the fact that a GA derivative with low affinity for Grp94 efficiently depleted ErbB2 protein in treated cells. To localize the specific region of ErbB2 that confers GA sensitivity, we made truncated receptors with progressive deletions of the cytoplasmic domain and tested the GA sensitivity of these molecules. We found that ErbB2 constructs containing an intact kinase domain retained GA sensitivity, whereas those lacking the kinase domain (ErbB2/DK) lost responsiveness to GA completely. Hsp90 co-immunoprecipitated with all ErbB2 constructs that were sensitive to GA, but not with ErbB2/DK or ErbB1. Both tyrosine-phosphorylated and non-phosphorylated ErbB2 proteins were similarly sensitive to GA, as was a kinase-dead ErbB2 mutant. These data suggest that Hsp90 uniquely stabilizes ErbB2 via interaction with its kinase domain and that GA stimulates ErbB2 degradation secondary to disruption of ErbB2/Hsp90 association.     

A Grp on the Hsp90 mechanism

In a recent issue of Molecular Cell, Dollins et al. (2007) present the crystal structure of Grp94, which highlights the similarity between Grp94 and Hsp90 and provides insight into the resting state of Grp94 and potentially other Hsp90 family members.     

Identification of novel quaternary domain interactions in the Hsp90 chaperone, GRP94

The structural basis for the coupling of ATP binding and hydrolysis to chaperone activity remains a central question in Hsp90 biology. By analogy to MutL, ATP binding to Hsp90 is thought to promote intramolecular N-terminal dimerization, yielding a molecular clamp functioning in substrate protein activation. Though observed in studies with recombinant domains, whether such quaternary states are present in native Hsp90s is unknown. In this study, native subunit interactions in GRP94, the endoplasmic reticulum Hsp90, were analyzed using chemical cross-linking in conjunction with tandem mass spectrometry. We report the identification of two distinct intermolecular interaction sites. Consistent with previous studies, one site comprises the C-terminal dimerization domain. The remaining site represents a novel intermolecular contact between the N-terminal and middle (M) domains of opposing subunits. This N+M domain interaction was present in the nucleotide-empty, ADP-, ATP-, or geldanamycin-bound states and could be selectively disrupted upon addition of synthetic geldanamycin dimers. These results identify a compact, intertwined quaternary conformation of native GRP94 and suggest that intersubunit N+M interactions are integral to the structural biology of Hsp90.     

Dynamics of the regulation of Hsp90 by the co-chaperone Sti1

In eukaryotic cells, Hsp90 chaperones assist late folding steps of many regulatory protein clients by a complex ATPase cycle. Binding of clients to Hsp90 requires prior interaction with Hsp70 and a transfer reaction that is mediated by the co-chaperone Sti1/Hop. Sti1 furthers client transfer by inhibiting Hsp90's ATPase activity. To better understand how Sti1 prepares Hsp90 for client acceptance, we characterized the interacting domains and analysed how Hsp90 and Sti1 mutually influence their conformational dynamics using hydrogen exchange mass spectrometry. Sti1 stabilizes several regions in all three domains of Hsp90 and slows down dissociation of the Hsp90 dimer. Our data suggest that Sti1 inhibits Hsp90's ATPase activity by preventing N-terminal dimerization and docking of the N-terminal domain with the middle domain. Using crosslinking and mass spectrometry we identified Sti1 segments, which are in close vicinity of the N-terminal domain of Hsp90. We found that the length of the linker between C-terminal dimerization domain and the C-terminal MEEVD motif is important for Sti1 association rates and propose a kinetic model for Sti1 binding to Hsp90.     

Different Poses for Ligand and Chaperone in Inhibitor-Bound Hsp90 and GRP94: Implications for Paralog-Specific Drug Design

Hsp90 chaperones contain an N-terminal ATP binding site that has been effectively targeted by competitive inhibitors. Despite the myriad of inhibitors, none to date have been designed to bind specifically to just one of the four mammalian Hsp90 paralogs, which are cytoplasmic Hsp90α and β, endoplasmic reticulum GRP94, and mitochondrial Trap-1. Given that each of the Hsp90 paralogs is responsible for chaperoning a distinct set of client proteins, specific targeting of one Hsp90 paralog may result in higher efficacy and therapeutic control. Specific inhibitors may also help elucidate the biochemical roles of each Hsp90 paralog. Here, we present side-by-side comparisons of the structures of yeast Hsp90 and mammalian GRP94, bound to the pan-Hsp90 inhibitors geldanamycin (Gdm) and radamide. These structures reveal paralog-specific differences in the Hsp90 and GRP94 conformations in response to Gdm binding. We also report significant variation in the pose and disparate binding affinities for the Gdm-radicicol chimera radamide when bound to the two paralogs, which may be exploited in the design of paralog-specific inhibitors.     

Primate chaperones Hsc70 (constitutive) and Hsp70 (induced) differ functionally in supporting growth and prion propagation in Saccharomyces cerevisiae

Hsp70's are highly conserved essential protein chaperones that assist protein folding and prevent protein aggregation. They have modular structures consisting of ATPase, substrate-binding, and C-terminal domains. Substrate binding and release is regulated by ATP hydrolysis and nucleotide exchange, which in turn are regulated by cochaperones. Eukaryotes have constitutive (Hsc70) and stress-inducible (iHsp70) isoforms, but their functions have not been systematically compared. Using a yeast system to evaluate heterologous Hsp70's we find that primate Hsc70 supported growth but iHsp70 did not. Plant Hsc70 and iHsp70 counterparts behaved similarly, implying evolutionary conservation of this distinction. Swapping yeast and primate Hsp70 domains showed that (i) the Hsc70-iHsp70 distinction resided in the ATPase domain, (ii) substrate-binding domains of Hsp70's within and across species functioned similarly regarding growth, (iii) C-terminal domain function was important for growth, and (iv) Hsp70 functions important for cell growth and prion propagation were separable. Enzymatic analysis uncovered a correlation between substrate affinity and prion phenotype and showed that ATPase and protein-folding activities were generally similar. Our data support a view that intrinsic activities of Hsp70 isoforms are comparable, and functional differences in vivo lie mainly in complex interactions of Hsp70 with cochaperones.