The Co-chaperone Sba1 connects the ATPase reaction of Hsp90 to the progression of the chaperone cycle

The molecular chaperone Hsp90 mediates the ATP-dependent activation of a large number of proteins involved in signal transduction. During this process, Hsp90 was found to associate transiently with several accessory factors, such as p23/Sba1, Hop/Sti1, and prolyl isomerases. It has been shown that ATP hydrolysis triggers conformational changes within Hsp90, which in turn are thought to mediate conformational changes in the substrate proteins, thereby causing their activation. The specific role of the partner proteins in this process is unknown. Using proteins from Saccharomyces cerevisiae, we characterized the interaction of Hsp90 with its partner protein p23/Sba1. Our results show that the nucleotide-dependent N-terminal dimerization of Hsp90 is necessary for the binding of Sba1 to Hsp90 with an affinity in the nanomolar range. Two Sba1 molecules were found to bind per Hsp90 dimer. Sba1 binding to Hsp90 resulted in a decreased ATPase activity, presumably by trapping the hydrolysis state of Hsp90ATP. Ternary complexes of Hsp90Sba1 could be formed with the prolyl isomerase Cpr6, but not with Sti1. Based on these findings, we propose a model that correlates the ordered assembly of the Hsp90 co-chaperones with distinct steps of the ATP hydrolysis reaction during the chaperone cycle.     

Substrate transfer from the chaperone Hsp70 to Hsp90

Hsp90 is an essential chaperone protein in the cytosol of eukaryotic cells. It cooperates with the chaperone Hsp70 in defined complexes mediated by the adaptor protein Hop (Sti1 in yeast). These Hsp70/Hsp90 chaperone complexes play a major role in the folding and maturation of key regulatory proteins in eukaryotes. Understanding how non-native client proteins are transferred from one chaperone to the other in these complexes is of central importance. Here, we analyzed the molecular mechanism of this reaction using luciferase as a substrate protein. Our experiments define a pathway for luciferase folding in the Hsp70/Hsp90 chaperone system. They demonstrate that Hsp70 is a potent capture device for unfolded protein while Hsp90 is not very efficient in this reaction. When Hsp90 is absent, in contrast to the in vivo situation, Hsp70 together with the two effector proteins Ydj1 and Sti1 exhibits chaperone activity towards luciferase. In the presence of the complete chaperone system, Hsp90 exhibits a specific positive effect only in the presence of Ydj1. If this factor is absent, the transferred luciferase is trapped on Hsp90 in an inactive conformation. Interestingly, identical results were observed for the yeast and the human chaperone systems although the regulatory function of human Hop is completely different from that of yeast Sti1.     

Cross-monomer substrate contacts reposition the Hsp90 N-terminal domain and prime the chaperone activity

The ubiquitous molecular chaperone Hsp90 plays a critical role in substrate protein folding and maintenance, but the functional mechanism has been difficult to elucidate. In previous work, a model Hsp90 substrate revealed an activation process in which substrate binding accelerates a large open/closed conformational change required for ATP hydrolysis by Hsp90. While this could serve as an elegant mechanism for conserving ATP usage for productive interactions on the substrate, the structural origin of substrate-catalyzed Hsp90 conformational changes is unknown. Here, we find that substrate binding affects an intrinsically unfavorable rotation of the Hsp90 N-terminal domain (NTD) relative to the middle domain (MD) that is required for closure. We identify an MD substrate binding region on the interior cleft of the Hsp90 dimer and show that a secondary set of substrate contacts drives an NTD orientation change on the opposite monomer. These results suggest an Hsp90 activation mechanism in which cross-monomer contacts mediated by a partially structured substrate prime the chaperone for its functional activity.     

Regulation of the Hsp90 system

Hsp90 is a highly conserved and abundant chaperone. It participates in essential cellular activities by supporting the maturation process of its client proteins, many of which are protein kinases and steroid receptors. Client processing is achieved via extensive conformational changes within the dimeric chaperone. This requires an ATP hydrolysis activity that is controlled by auto-inhibitory mechanisms and several structurally diverse cofactors. Especially the client-specificity of Hsp90 depends on client-specific cofactors, which can adapt Hsp90's activities to the client requirements at different conditions and in different cell types. Additionally, post-translational modifications can influence almost every aspect of Hsp90's interactions and activities. In this review, we present these regulatory principles, discuss the factors that have an impact on Hsp90's function and elaborate the mechanisms that are responsible for regulating the Hsp90 machinery.     

Dancing with the Diva: Hsp90–Client Interactions

The molecular chaperone Hsp90 is involved in the folding, maturation, and degradation of a large number structurally and sequentially unrelated clients, often connected to serious diseases. Elucidating the principles of how Hsp90 recognizes this large variety of substrates is essential for comprehending the mechanism of this chaperone machinery, as well as it is a prerequisite for the design of client specific drugs targeting Hsp90. Here, we discuss the recent progress in understanding the substrate recognition principles of Hsp90 and its implications for the role of Hsp90 in the lifecycle of proteins. Hsp90 acts downstream of the chaperone Hsp70, which exposes its substrate to a short and highly hydrophobic cleft. The subsequently acting Hsp90 has an extended client-binding interface that enables a large number of low-affinity contacts. Structural studies show interaction modes of Hsp90 with the intrinsically disordered Alzheimer's disease-causing protein Tau, the kinase Cdk4 in a partially unfolded state and the folded ligand-binding domain of a steroid receptor. Comparing the features shared by these different proteins provides a picture of the substrate-binding principles of Hsp90.     

Evidence for chaperone heterocomplexes containing both Hsp90 and VCP

With assistance from co-chaperone partner proteins, Hsp90 plays an essential positive role in supporting the structure and function of numerous client proteins in vivo. Hsp90's co-chaperone partnerships are believed to regulate and/or target its function. Here we describe associations between Hsp90 chaperone machinery and another chaperone, the 97-kDa valosin-containing protein VCP. Coimmunoadsorption assays indicate that VCP occurs in one or more native heterocomplexes containing Hsp90 and the Hsp90 partner proteins Cdc37, FKBP52, and p23. Functional characterizations indicate that VCP is not an Hsp90 substrate, but rather demonstrate the biochemical hallmarks of an Hsp90 co-chaperone. Potential roles for a collaboration between for Hsp90 and VCP are discussed.     

Hsp90伴侣复合体装配及对类固醇受体调节

真核细胞中近100种蛋白质都受Hsp90的调节。这些蛋白质多与信号转导作用有关,它们与Hsp90一起进入一个以Hsp90/Hsp70为主的伴侣复合体,在复合体内完成信号转导作用。Hsp90除了和蛋白质的伴侣位点结合以外,还在其他位点与辅助因子连接,这是Hsp90能与蛋白质及辅助因子组装成复合体,并进而调节其信号作用的结构基础。类固醇受体等蛋白质的信号转导作用是在Hsp70、Hsp90为基础的5种蛋白质(Hsp90,Hsp70,Hop,Hsp40和p23)组成的复合体中进行的。这个系统可以帮助理解在真核细胞中,Hsp70和Hsp90怎样联合作用,改变底物蛋白构象,以及怎样应答信号作用。     

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.     

More than folding: localized functions of cytosolic chaperones

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

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.     

A comparison of Hsp90alpha and Hsp90beta interactions with cochaperones and substrates.

Hsp90 chaperone complexes function in assembly, folding, and activation of numerous substrates. The 2 vertebrate homologues encoded by the genes hsp90a and hsp90b are differentially expressed in embryonic and adult tissues and during stress; however, it is not known whether they possess identical functional activities in chaperone complexes. This question was addressed by examining potential differences between the Hsp90 isoforms with respect to both cochaperone and substrate interactions. Epitope-tagged proteins were expressed in mammalian cells or Xenopus oocytes and subjected to immunoprecipitation with an array of cochaperones. Both isoforms were shown to participate equally in multichaperone complexes, and no significant differences in cochaperone distribution were observed. The substrates Raf-1, HSF1, Cdc37, and MEK1 interacted with both Hsp90alpha and Hsp90beta, and the relative patterns of these interactions were not affected by heat shock. The substrate kinases c-Src, CKIIB, A-raf, and Erk interacted with both isoforms; however, significantly more Hsp90alpha was recovered after heat shock. The data demonstrate that Hsp90alpha and Hsp90beta exhibit similar interactions with cochaperones, but significantly different behaviors with respect to substrate interactions under stress conditions. These results reveal both functional similarities and key functional differences in the individual members of this protein family.     

Modulation of heme/substrate binding cleft of neuronal nitric-oxide synthase (nNOS) regulates binding of Hsp90 and Hsp70 proteins and nNOS ubiquitination

Like other nitric-oxide synthase (NOS) enzymes, neuronal NOS (nNOS) turnover and activity are regulated by the Hsp90/Hsp70-based chaperone machinery, which regulates signaling proteins by modulating ligand binding clefts (Pratt, W. B., Morishima, Y., and Osawa, Y. (2008) J. Biol. Chem. 283, 22885-22889). We have previously shown that nNOS turnover is due to Hsp70/CHIP-dependent ubiquitination and proteasomal degradation. In this work, we use an intracellular cross-linking approach to study both chaperone binding and nNOS ubiquitination in intact HEK293 cells. Treatment of cells with N(G)-nitro-L-arginine, a slowly reversible competitive inhibitor that stabilizes nNOS, decreases both nNOS ubiquitination and binding of Hsp90, Hsp70, and CHIP. Treatment with the calcium ionophore A23187, which increases Ca(2+)-calmodulin binding to nNOS, increases nNOS ubiquitination and binding of Hsp90, Hsp70, and CHIP in a manner that is specific for changes in the heme/substrate binding cleft. Both Hsp90 and Hsp70 are bound to the expressed nNOS oxygenase domain, which contains the heme/substrate binding cleft, but not to the reductase domain, and binding is increased to an expressed fragment containing both the oxygenase domain and the calmodulin binding site. Overexpression of Hsp70 promotes nNOS ubiquitination and decreases nNOS protein, and overexpression of Hsp90 inhibits nNOS ubiquitination and increases nNOS protein, showing the opposing effects of the two chaperones as they participate in nNOS quality control in the cell. These observations support the notion that changes in the state of the heme/substrate binding cleft affect chaperone binding and thus nNOS ubiquitination.     

Evolution and function of diverse Hsp90 homologs and cochaperone proteins

Members of the Hsp90 molecular chaperone family are found in the cytosol, ER, mitochondria and chloroplasts of eukaryotic cells, as well as in bacteria. These diverse family members cooperate with other proteins, such as the molecular chaperone Hsp70, to mediate protein folding, activation and assembly into multiprotein complexes. All examined Hsp90 homologs exhibit similar ATPase rates and undergo similar conformational changes. One of the key differences is that cytosolic Hsp90 interacts with a large number of cochaperones that regulate the ATPase activity of Hsp90 or have other functions, such as targeting clients to Hsp90. Diverse Hsp90 homologs appear to chaperone different types of client proteins. This difference may reflect either the pool of clients requiring Hsp90 function or the requirement for cochaperones to target clients to Hsp90. This review discusses known functions, similarities and differences between Hsp90 family members and how cochaperones are known to affect these functions. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

The mammalian CHORD-containing protein melusin is a stress response protein interacting with Hsp90 and Sgt1

Melusin is a mammalian muscle specific CHORD containing protein capable of activating signal transduction pathways leading to cardiomyocytes hypertrophy in response to mechanical stress. To define melusin function we searched for molecular partners possibly involved in melusin dependent signal transduction. Here we show that melusin and heat shock proteins are co-regulated. Moreover, melusin directly binds to Hsp90, a ubiquitous chaperone involved in regulating several signaling pathways. In addition, melusin interacts with Sgt1, an Hsp90 binding molecule. Melusin does not behave as an Hsp90 substrate but rather as a chaperone capable to protect citrate synthase from heat induced aggregation. These results describe melusin as a new component of the Hsp90 chaperone machinery.     

GERp95 belongs to a family of signal-transducing proteins and requires Hsp90 activity for stability and Golgi localization.

GERp95 (Golgi-endoplasmic reticulum protein 95 kDa) is part of a large family of highly conserved proteins found in all metazoans and the fission yeast Schizosaccharomyces pombe. Genetic studies suggest that homologs of GERp95 are components of signaling pathways that regulate cellular differentiation, development, and RNA interference. However, the precise molecular functions of these proteins remain unknown. Genetic analysis of GERp95 homologs has been complicated by the presence of multiple genes with overlapping functions in most organisms. Binding partners for members of this protein family have not been identified. The purpose of this study was to identify proteins that associate with GERp95. Glutathione S-transferase-GERp95 fusions were expressed in transfected cells, and proteins that bound to GERp95 were co-purified using glutathione-agarose beads. The amino-terminal region of GERp95 was found to interact with the specialized chaperone Hsp90 and a number of its cognate binding proteins. Inhibition of Hsp90 activity with geldanamycin or radicicol resulted in rapid degradation of newly synthesized GERp95. The membrane-associated pool of GERp95 was not bound to Hsp90, although activity of this chaperone was required for stable association of GERp95 with the Golgi in normal rat kidney cells. These results indicate that GERp95 engages an Hsp90 chaperone complex prior to association with intracellular membranes.     

The phosphatase Ppt1 is a dedicated regulator of the molecular chaperone Hsp90

Ppt1 is the yeast member of a novel family of protein phosphatases, which is characterized by the presence of a tetratricopeptide repeat (TPR) domain. Ppt1 is known to bind to Hsp90, a molecular chaperone that performs essential functions in the folding and activation of a large number of client proteins. The function of Ppt1 in the Hsp90 chaperone cycle remained unknown. Here, we analyzed the function of Ppt1 in vivo and in vitro. We show that purified Ppt1 specifically dephosphorylates Hsp90. This activity requires Hsp90 to be directly attached to Ppt1 via its TPR domain. Deletion of the ppt1 gene leads to hyperphosphorylation of Hsp90 in vivo and an apparent decrease in the efficiency of the Hsp90 chaperone system. Interestingly, several Hsp90 client proteins were affected in a distinct manner. Our findings indicate that the Hsp90 multichaperone cycle is more complex than was previously thought. Besides its regulation via the Hsp90 ATPase activity and the sequential binding and release of cochaperones, with Ppt1, a specific phosphatase exists, which positively modulates the maturation of Hsp90 client proteins.     

Cpr6 and Cpr7, two closely related Hsp90-associated immunophilins from Saccharomyces cerevisiae, differ in their functional properties

Hsp90 is an abundant cytosolic molecular chaperone. It controls the folding of target proteins including steroid hormone receptors and kinases in complex with several partner proteins. Prominent members of this protein family are large peptidyl prolyl cis/trans isomerases (PPIases), which catalyze the cis/trans isomerization of prolyl peptide bonds in proteins and possess chaperone activity. In Saccharomyces cerevisiae, two closely related large Hsp90-associated PPIases, Cpr6 and Cpr7, exist. We show here that these homologous proteins bind with comparable affinity to Hsp90 but exhibit significant structural and functional differences. Cpr6 is more stable than Cpr7 against thermal denaturation and displays an up to 100-fold higher PPIase activity. In contrast, the chaperone activity of Cpr6 is much lower than that of Cpr7. Based on these results we suggest that the two immunophilins perform overlapping but not identical tasks in the Hsp90 chaperone cycle.     

Molecular chaperone Hsp90 stabilizes Pih1/Nop17 to maintain R2TP complex activity that regulates snoRNA accumulation

Hsp90 is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes. In this study, we identify a function for the chaperone in RNA processing and maintenance. This functionality of Hsp90 involves two recently identified interactors of the chaperone: Tah1 and Pih1/Nop17. Tah1 is a small protein containing tetratricopeptide repeats, whereas Pih1 is found to be an unstable protein. Tah1 and Pih1 bind to the essential helicases Rvb1 and Rvb2 to form the R2TP complex, which we demonstrate is required for the correct accumulation of box C/D small nucleolar ribonucleoproteins. Together with the Tah1 cofactor, Hsp90 functions to stabilize Pih1. As a consequence, the chaperone is shown to affect box C/D accumulation and maintenance, especially under stress conditions. Hsp90 and R2TP proteins are also involved in the proper accumulation of box H/ACA small nucleolar RNAs.     

The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins

To maintain quality control in cells, mechanisms distinguish among improperly folded peptides, mature and functional proteins, and proteins to be targeted for degradation. The molecular chaperones, including heat-shock protein Hsp90, have the ability to recognize misfolded proteins and assist in their conversion to a functional conformation. Disruption of Hsp90 heterocomplexes by the Hsp90 inhibitor geldanamycin leads to substrate degradation through the ubiquitin-proteasome pathway, implicating this system in protein triage decisions. We previously identified CHIP (carboxyl terminus of Hsc70-interacting protein) to be an interaction partner of Hsc70 (ref. 4). CHIP also interacts directly with a tetratricopeptide repeat acceptor site of Hsp90, incorporating into Hsp90 heterocomplexes and eliciting release of the regulatory cofactor p23. Here we show that CHIP abolishes the steroid-binding activity and transactivation potential of the glucocorticoid receptor, a well-characterized Hsp90 substrate, even though it has little effect on its synthesis. Instead, CHIP induces ubiquitylation of the glucocorticoid receptor and degradation through the proteasome. By remodelling Hsp90 heterocomplexes to favour substrate degradation, CHIP modulates protein triage decisions that regulate the balance between protein folding and degradation for chaperone substrates.     

Cyclic lipopeptide antibiotics bind to the N-terminal domain of the prokaryotic Hsp90 to inhibit the chaperone activity

Chemical arrays were employed to screen ligands for HtpG, the prokaryotic homologue of Hsp (heat-shock protein) 90. We found that colistins and the closely related polymyxin B interact physically with HtpG. They bind to the N-terminal domain of HtpG specifically without affecting its ATPase activity. The interaction caused inhibition of chaperone function of HtpG that suppresses thermal aggregation of substrate proteins. Further studies were performed with one of these cyclic lipopeptide antibiotics, colistin sulfate salt. It inhibited the chaperone function of the N-terminal domain of HtpG. However, it inhibited neither the chaperone function of the middle domain of HtpG nor that of other molecular chaperones such as DnaK, the prokaryotic homologue of Hsp70, and small Hsp. The addition of colistin sulfate salt increased surface hydrophobicity of the N-terminal domain of HtpG and induced oligomerization of HtpG and its N-terminal domain. These structural changes are discussed in relation to the inhibition of the chaperone function.