Structural basis for recruitment of the ATPase activator Aha1 to the Hsp90 chaperone machinery

Hsp90 is a molecular chaperone essential for the activation and assembly of many key eukaryotic signalling and regulatory proteins. Hsp90 is assisted and regulated by co-chaperones that participate in an ordered series of dynamic multiprotein complexes, linked to Hsp90s conformationally coupled ATPase cycle. The co-chaperones Aha1 and Hch1 bind to Hsp90 and stimulate its ATPase activity. Biochemical analysis shows that this activity is dependent on the N-terminal domain of Aha1, which interacts with the central segment of Hsp90. The structural basis for this interaction is revealed by the crystal structure of the N-terminal domain (1-153) of Aha1 (equivalent to the whole of Hch1) in complex with the middle segment of Hsp90 (273-530). Structural analysis and mutagenesis show that binding of N-Aha1 promotes a conformational switch in the middle-segment catalytic loop (370-390) of Hsp90 that releases the catalytic Arg 380 and enables its interaction with ATP in the N-terminal nucleotide-binding domain of the chaperone.     

Aha1 binds to the middle domain of Hsp90, contributes to client protein activation,and stimulates the ATPase activity of the molecular chaperone

The ATP-dependent molecular chaperone Hsp90 is an essential and abundant stress protein in the eukaryotic cytosol that cooperates with a cohort of cofactors/cochaperones to fulfill its cellular tasks. We have identified Aha1 (activator of Hsp90 ATPase) and its relative Hch1 (high copy Hsp90 suppressor) as binding partners of Hsp90 in Saccharomyces cerevisiae. By using genetic and biochemical approaches, the middle domain of Hsp90 (amino acids 272-617) was found to mediate the interaction with Aha1 and Hch1. Data base searches revealed that homologues of Aha1 are conserved from yeast to man, whereas Hch1 was found to be restricted to lower eukaryotes like S. cerevisiae and Candida albicans. In experiments with purified proteins, Aha1 but not Hch1 stimulated the intrinsic ATPase activity of Hsp90 5-fold. To establish their cellular role further, we deleted the genes encoding Aha1 and Hch1 in S. cerevisiae. In vivo experiments demonstrated that Aha1 and Hch1 contributed to efficient activation of the heterologous Hsp90 client protein v-Src. Moreover, Aha1 and Hch1 became crucial for cell viability under non-optimal growth conditions when Hsp90 levels are limiting. Thus, our results identify a novel type of cofactor involved in the regulation of the molecular chaperone Hsp90.     

Co-chaperone regulation of conformational switching in the Hsp90 ATPase cycle

ATP hydrolysis by the Hsp90 molecular chaperone requires a connected set of conformational switches triggered by ATP binding to the N-terminal domain in the Hsp90 dimer. Central to this is a segment of the structure, which closes like a "lid" over bound ATP, promoting N-terminal dimerization and assembly of a competent active site. Hsp90 mutants that influence these conformational switches have strong effects on ATPase activity. ATPase activity is specifically regulated by Hsp90 co-chaperones, which directly influence the conformational switches. Here we have analyzed the effect of Hsp90 mutations on binding (using isothermal titration calorimetry and difference circular dichroism) and ATPase regulation by the co-chaperones Aha1, Sti1 (Hop), and Sba1 (p23). The ability of Sti1 to bind Hsp90 and arrest its ATPase activity was not affected by any of the mutants screened. Sba1 bound in the presence of AMPPNP to wild-type and ATPase hyperactive mutants with similar affinity but only very weakly to hypoactive mutants despite their wild-type ATP affinity. Unexpectedly, in all cases Sba1 bound to Hsp90 with a 1:2 molar stoichiometry. Aha1 binding to mutants was similar to wild-type, but the -fold activation of their ATPase varied substantially between mutants. Analysis of complex formation with co-chaperone mixtures showed Aha1 and p50cdc37 able to bind Hsp90 simultaneously but without direct interaction. Sba1 and p50cdc37 bound independently to Hsp90-AMPPNP but not together. These data indicated that Sba1 and Aha1 regulate Hsp90 by influencing the conformational state of the "ATP lid" and consequent N-terminal dimerization, whereas Sti1 does not.     

A Mutation in the Catalytic Loop of Hsp90 Specifically Impairs ATPase Stimulation by Aha1p,But Not Hch1p

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays a central role in maintaining cellular homeostasis by facilitating activation of a large number of client proteins. ATP-dependent client activation by Hsp90 is tightly regulated by a host of co-chaperone proteins that control progression through the activation cycle. ATPase stimulation of Hsp90 by Aha1p requires a conserved RKxK motif that interacts with the catalytic loop of Hsp90. In this study, we explore the role of this RKxK motif in the biological and biochemical properties of Hch1p. We found that this motif is required for Hch1p-mediated ATPase stimulation in vitro, but mutations that block stimulation do not impair the action of Hch1p in vivo. This suggests that the biological function of Hch1p is not directly linked to ATPase stimulation. Moreover, a mutation in the catalytic loop of Hsp90 specifically impairs ATPase stimulation by Aha1p but not by Hch1p. Our work here suggests that both Hch1p and Aha1p regulate Hsp90 function through interaction with the catalytic loop but do so in different ways.     

Characterization of the interaction of Aha1 with components of the Hsp90 chaperone machine and client proteins

The activator of Hsp90 ATPase, Aha1, is an Hsp90 co-chaperone that has been suggested to act as a general stimulator of Hsp90 function. In this report, we have characterized the interaction of Aha1 with Hsp90 and its co-chaperones in rabbit reticulocyte lysate (RRL) and in HeLa cell extracts. Complexes formed by Aha1 with Hsp90 in RRL were stabilized by molybdate and contained the co-chaperones FKBP52 and p23/Sba1, but lacked HOP/Sti1 and Cdc37. Aha1 complexes isolated from HeLa cell extracts also contained Hsp70 and DNAJA1. Over-expression of Aha1 has been reported to stimulate the activity of v-Src and steroid hormone receptors ectopically expressed in yeast, however, no interaction between Aha1 and nascent v-Src or the progesterone receptor could be detected in RRL. Contrary to expectations, over-expression of Aha1 also inhibited the rate of Hsp90-dependent refolding of denatured luciferase. A number of potential client proteins that specifically associated with Aha1 were identified by liquid chromatography/ tandem mass spectrometry (LC-MS/MS) and verified by Western blotting. The proteins identified suggest that Aha1 may play roles in modulating RNA splicing and DNA repair, in addition to other cellular processes.     

Activation of the ATPase activity of hsp90 by the stress-regulated cochaperone aha1

Client protein activation by Hsp90 involves a plethora of cochaperones whose roles are poorly defined. A ubiquitous family of stress-regulated proteins have been identified (Aha1, activator of Hsp90 ATPase) that bind directly to Hsp90 and are required for the in vivo Hsp90-dependent activation of clients such as v-Src, implicating them as cochaperones of the Hsp90 system. In vitro, Aha1 and its shorter homolog, Hch1, stimulate the inherent ATPase activity of yeast and human Hsp90. The identification of these Hsp90 cochaperone activators adds to the complex roles of cochaperones in regulating the ATPase-coupled conformational changes of the Hsp90 chaperone cycle.     

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

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

Enforced N-domain proximity stimulates Hsp90 ATPase activity and is compatible with function in vivo

Hsp90 populates distinct open and closed conformations mediated by transient N-terminal dimerization. To investigate the mechanistic role of these large conformational changes, we designed Hsp90 with an N-terminal coiled-coil to clamp the termini together and enforce N-domain proximity. Biophysical analyses demonstrate that the coiled-coil effectively maintains N-domain proximity in the absence of ATP, a condition that favors the open state of Hsp90. Enforcing N-domain proximity results in increased ATPase activity, indicating that N-terminal dimerization is a rate-limiting step that is sped-up with the coiled-coil due to increased effective N-domain concentration. The relative difference in ATPase activity between coil-Hsp90 and wt was reduced in the presence of both an ATPase activating (Aha1) and an inhibiting (Sba1) co-chaperone. As both of these co-chaperones bind preferentially to N-terminally dimerized Hsp90, the buffering effect of these co-chaperones demonstrates the biochemical relevance of Hsp90 conformational properties in addition to N-terminal dimerization. Enforcing N-domain proximity is compatible with viability in yeast, underlining the mechanistic relevance of Hsp90 conformational changes that are less dramatic than the transition between fully open and closed.     

Cdc37-Hsp90 complexes are responsive to nucleotide-induced conformational changes and binding of further cofactors

Hsp90 is an ATP-dependent molecular chaperone, which facilitates the activation and stabilization of hundreds of client proteins in cooperation with a defined set of cofactors. Many client proteins are protein kinases, which are activated and stabilized by Hsp90 in cooperation with the kinase-specific co-chaperone Cdc37. Other Hsp90 co-chaperones, like the ATPase activator Aha1, also are implicated in kinase activation, and it is not yet clear how Cdc37 is integrated into Hsp90 co-chaperone complexes. Here, we studied the interaction between Cdc37, Hsp90, and other Hsp90 co-chaperones from the nematode Caenorhabditis elegans. Nematode Cdc37 binds with high affinity to Hsp90 and strongly inhibits the ATPase activity. In contrast to the human Hsp90 system, we observed binding of Cdc37 to open and closed Hsp90 conformations, potentially reflecting two different binding modes. Using a novel ultracentrifugation setup, which allows accurate analysis of multifactorial protein complexes, we show that cooperative and competitive interactions exist between other co-chaperones and Cdc37-Hsp90 complexes in the C. elegans system. We observed strong competitive interactions between Cdc37 and the co-chaperones p23 and Sti1, whereas the binding of the phosphatase Pph5 and the ATPase activator Aha1 to Cdc37-Hsp90 complexes is possible. The ternary Aha1-Cdc37-Hsp90 complex is disrupted by the nucleotide-induced closing reaction at the N terminus of Hsp90. This implies a carefully regulated exchange process of cofactors during the chaperoning of kinase clients by Hsp90.     

The Co-Chaperone Hch1 Regulates Hsp90 Function Differently than Its Homologue Aha1 and Confers Sensitivity to Yeast to the Hsp90 Inhibitor NVP-AUY922

Hsp90 is a dimeric ATPase responsible for the activation or maturation of a specific set of substrate proteins termed ‘clients’. This molecular chaperone acts in the context of a structurally dynamic and highly regulated cycle involving ATP, co-chaperone proteins and clients. Co-chaperone proteins regulate conformational transitions that may be impaired in mutant forms of Hsp90. We report here that the in vivo impairment of commonly studied Hsp90 variants harbouring the G313S or A587T mutation are exacerbated by the co-chaperone Hch1p. Deletion of HCH1, but not AHA1, mitigates the temperature sensitive phenotype and high sensitivity to Hsp90 inhibitor drugs observed in Saccharomyces cerevisiae that express either of these two Hsp90 variants. Moreover, the deletion of HCH1 results in high resistance to Hsp90 inhibitors in yeast that express wildtype Hsp90. Conversely, the overexpression of Hch1p greatly increases sensitivity to Hsp90 inhibition in yeast expressing wildtype Hsp90. We conclude that despite the similarity between these two co-chaperones, Hch1p and Aha1p regulate Hsp90 function in distinct ways and likely independent of their roles as ATPase stimulators. We further conclude that Hch1p plays a critical role in regulating Hsp90 inhibitor drug sensitivity in yeast.     

Threonine 22 phosphorylation attenuates Hsp90 interaction with cochaperones and affects its chaperone activity

Heat shock protein 90 (Hsp90) is an essential molecular chaperone whose activity is regulated not only by cochaperones but also by distinct posttranslational modifications. We report here that casein kinase 2 phosphorylates a conserved threonine residue (T22) in α helix-1 of the yeast Hsp90 N-domain both in?vitro and in?vivo. This α helix participates in?a hydrophobic interaction with the catalytic loop in Hsp90's middle domain, helping to stabilize the chaperone's ATPase-competent state. Phosphomimetic mutation of this residue alters Hsp90 ATPase activity and chaperone function and impacts interaction with the cochaperones Aha1 and Cdc37. Overexpression of Aha1 stimulates the ATPase activity, restores cochaperone interactions, and compensates for the functional defects of these Hsp90 mutants.     

Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)-domain co-chaperones.

The in vivo function of the heat shock protein 90 (Hsp90) molecular chaperone is dependent on the binding and hydrolysis of ATP, and on interactions with a variety of co-chaperones containing tetratricopeptide repeat (TPR) domains. We have now analysed the interaction of the yeast TPR-domain co-chaperones Sti1 and Cpr6 with yeast Hsp90 by isothermal titration calorimetry, circular dichroism spectroscopy and analytical ultracentrifugation, and determined the effect of their binding on the inherent ATPase activity of Hsp90. Sti1 and Cpr6 both bind with sub-micromolar affinity, with Sti1 binding accompanied by a large conformational change. Two co-chaperone molecules bind per Hsp90 dimer, and Sti1 itself is found to be a dimer in free solution. The inherent ATPase activity of Hsp90 is completely inhibited by binding of Sti1, but is not affected by Cpr6, although Cpr6 can reactivate the ATPase activity by displacing Sti1 from Hsp90. Bound Sti1 makes direct contact with, and blocks access to the ATP-binding site in the N-terminal domain of Hsp90. These results reveal an important role for TPR-domain co-chaperones as regulators of the ATPase activity of Hsp90, showing that the ATP-dependent step in Hsp90-mediated protein folding occurs after the binding of the folding client protein, and suggesting that ATP hydrolysis triggers client-protein release.     

Hsp90 inhibitors radicicol and geldanamycin have opposing effects on <Emphasis Type="Italic">Leishmania</Emphasis> Aha1-dependent proliferation

Hsp90 and its co-chaperones are essential for the medically important parasite Leishmania donovani, facilitating life cycle control and intracellular survival. Activity of Hsp90 is regulated by co-chaperones of the Aha1 and P23 families. In this paper, we studied the expression of L. donovani Aha1 in two life cycle stages, its interaction with Hsp90 and the phenotype of Aha1 null mutants during the insect stage and inside infected macrophages. This study provides a detailed in vitro analysis of the function of Aha1 in Leishmania parasites and the first instance of a reverse genetic analysis of Aha1 in a protozoan parasite. While Aha1 is non-essential under standard growth conditions and at elevated temperature, Aha1 protects against ethanol stress. However, both overexpression and lack of Aha1 affected parasite growth in the presence of the Hsp90 inhibitors radicicol (RAD) and geldanamycin (GA). Under RAD pressure, P23 and Aha1 act in an antagonistic way. By contrast, expression levels of both co-chaperones have similar effects under GA treatment, indicating different inhibition mechanisms by the two compounds. Aha1 is also secreted in virulence-enhancing exosomes. This may explain why the loss of Aha1 reduces the infectivity of L. donovani in ex vivo mouse macrophages, indicating a role during the intracellular mammalian stage.     

Dynamic Aha1 co‐chaperone binding to human Hsp90

Hsp90 is an essential chaperone that requires large allosteric changes to determine its ATPase activity and client binding. The co‐chaperone Aha1, which is the major ATPase stimulator in eukaryotes, is important for regulation of Hsp90's allosteric timing. Little is known, however, about the structure of the Hsp90/Aha1 complex. Here, we characterize the solution structure of unmodified human Hsp90/Aha1 complex using NMR spectroscopy. We show that the 214‐kDa complex forms by a two‐step binding mechanism and adopts multiple conformations in the absence of nucleotide. Aha1 induces structural changes near Hsp90's nucleotide‐binding site, providing a basis for its ATPase‐enhancing activity. Our data reveal important aspects of this pivotal chaperone/co‐chaperone interaction and emphasize the relevance of characterizing dynamic chaperone structures in solution.     

The Mechanism of Hsp90 regulation by the protein kinase-specific cochaperone p50(cdc37)

Recruitment of protein kinase clients to the Hsp90 chaperone involves the cochaperone p50(cdc37) acting as a scaffold, binding protein kinases via its N-terminal domain and Hsp90 via its C-terminal region. p50(cdc37) also has a regulatory activity, arresting Hsp90's ATPase cycle during client-protein loading. We have localized the binding site for p50(cdc37) to the N-terminal nucleotide binding domain of Hsp90 and determined the crystal structure of the Hsp90-p50(cdc37) core complex. Dimeric p50(cdc37) binds to surfaces of the Hsp90 N-domain implicated in ATP-dependent N-terminal dimerization and association with the middle segment of the chaperone. This interaction fixes the lid segment in an open conformation, inserts an arginine side chain into the ATP binding pocket to disable catalysis, and prevents trans-activating interaction of the N domains.     

Low Resolution Structural Studies Indicate that the Activator of Hsp90 ATPase 1 (Aha1) of Leishmania braziliensis Has an Elongated Shape Which Allows Its Interaction with Both N- and M-Domains of Hsp90

The Hsp90 molecular chaperone is essential for protein homeostasis and in the maturation of proteins involved with cell-cycle control. The low ATPase activity of Hsp90 is critical to drive its functional cycle, which is dependent on the Hsp90 cochaperones. The Activator of Hsp90 ATPase-1 (Aha1) is a protein formed by two domains, N- and C-terminal, that stimulates the Hsp90 ATPase activity by several folds. Although the relevance of Aha1 for Hsp90 functions has been proved, as well as its involvement in the desensitization to inhibitors of the Hsp90, the knowledge on its overall structure and behavior in solution is limited. In this work we present the functional and structural characterization of Leishmania braziliensis Aha1 (LbAha1). This protozoan is the causative agent of cutaneous and mucocutaneous leishmaniasis, a neglected disease. The recombinant LbAha1 behaves as an elongated monomer and is organized into two folded domains interconnected by a flexible linker. Functional experiments showed that LbAha1 interacts with L. braziliensis Hsp90 (LbHsp90) with micromolar dissociation constant in a stoichiometry of 2 LbAha1 to 1 LbHsp90 dimer and stimulates 10-fold the LbHsp90 ATPase activity showing positive cooperativity. Furthermore, the LbHsp90::LbAha1 complex is directed by enthalphy and opposed by entropy, probably due to the spatial freedom restrictions imposed by the proteins’ interactions. Small-angle X-ray scattering data allowed the reconstruction of low resolution models and rigid body simulations of LbAha1, indicating its mode of action on LbHsp90. Western blot experiments allowed Aha1 identification (as well as Hsp90) in three Leishmania species at two temperatures, suggesting that Aha1 is a cognate protein. All these data shed light on the LbAha1 mechanism of action, showing that it has structural dimensions and flexibility that allow interacting with both N-terminal and middle domains of the LbHsp90.     

Cns1 is an activator of the Ssa1 ATPase activity

Hsp90 is a key mediator in the folding process of a growing number of client proteins. The molecular chaperone cooperates with many co-chaperones and partner proteins to fulfill its task. In Saccharomyces cerevisiae, several co-chaperones of Hsp90 interact with Hsp90 via a tetratricopeptide repeat (TPR) domain. Here we show that one of these proteins, Cns1, binds both to Hsp90 and to the yeast Hsp70 protein Ssa1 with comparable affinities. This is reminiscent of Sti1, another TPR-containing co-chaperone. Unlike Sti1, Cns1 exhibits no influence on the ATPase of Hsp90. However, it activates the ATPase of Ssa1 up to 30-fold by accelerating the rate-limiting ATP hydrolysis step. This stimulating effect is mediated by the N-terminal TPR-containing part of Cns1, whereas the C-terminal part showed no effect. Competition experiments allow the conclusion that Hsp90 and Ssa1 compete for binding to the single TPR domain of Cns1. Taken together, Cns1 is a potent cochaperone of Ssa1. Our findings highlight the importance of the regulation of Hsp70 function in the context of the Hsp90 chaperone cycle.     

Aha1 Can Act as an Autonomous Chaperone to Prevent Aggregation of Stressed Proteins

Aha1 (activator of Hsp90 ATPase) stimulates the ATPase activity of the molecular chaperone Hsp90 to accelerate the conformational cycle during which client proteins attain their final shape. Thereby, Aha1 promotes effective folding of Hsp90-dependent clients such as steroid receptors and many kinases involved in cellular signaling. In our current study, we find that Aha1 plays a novel, additional role beyond regulating the Hsp90 ATP hydrolysis rate. We propose a new concept suggesting that Aha1 acts as an autonomous chaperone and associates with stress-denatured proteins to prevent them from aggregation similar to the chaperonin GroEL. Our study reveals that an N-terminal sequence of 22 amino acids, present in human but absent from yeast Aha1, is critical for this capability. However, in lieu of fostering their refolding, Aha1 allows ubiquitination of bound clients by the E3 ubiquitin ligase CHIP. Accordingly, Aha1 may promote disposal of folding defective proteins by the cellular protein quality control.     

Virtual prototyping study shows increased ATPase activity of Hsp90 to be the key determinant of cancer phenotype

Hsp90 is an ATP-dependent molecular chaperone that regulates key signaling proteins and thereby impacts cell growth and development. Chaperone cycle of Hsp90 is regulated by ATP binding and hydrolysis through its intrinsic ATPase activities, which is in turn modulated by interaction with its co-chaperones. Hsp90 ATPase activity varies in different organisms and is known to be increased in tumor cells. In this study we have quantitatively analyzed the impact of increasing Hsp90 ATPase activity on the activities of its clients through a virtual prototyping technology, which comprises a dynamic model of Hsp90 interaction with clients involved in proliferation pathways. Our studies highlight the importance of increased ATPase activity of Hsp90 in cancer cells as the key modulator for increased proliferation and survival. A tenfold increase in ATPase activity of Hsp90 often seen in cancer cells increases the levels of active client proteins such as Akt-1, Raf-1 and Cyclin D1 amongst others to about 12-, 8- and 186-folds respectively. Additionally we studied the effect of a competitive inhibitor of Hsp90 activity on the reduction in the client protein levels. Virtual prototyping experiments corroborate with findings that the drug has almost 10- to 100-fold higher affinity as indicated by a lower IC₅₀ value (30–100 nM) in tumor cells with higher ATPase activity. The results also indicate a 15- to 25-fold higher efficacy of the inhibitor in reducing client levels in tumor cells. This analysis provides mechanistic insights into the links between increased Hsp90 ATPase activity, tumor phenotype and the hypersensitivity of tumor Hsp90 to inhibition by ATP analogs.     

Biological and Structural Basis for Aha1 Regulation of Hsp90 ATPase Activity in Maintaining Proteostasis in the Human Disease Cystic Fibrosis

The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90. To elucidate the structural basis for ATPase stimulation of human Hsp90 by human Aha1, we have developed novel mass spectrometry approaches that demonstrate that the N- and C-terminal domains of Aha1 cooperatively bind across the dimer interface of Hsp90 to modulate the ATP hydrolysis cycle and client activity in vivo. Mutations in both the N- and C-terminal domains of Aha1 impair its ability to bind Hsp90 and stimulate its ATPase activity in vitro and impair in vivo the ability of the Hsp90 system to modulate the folding and trafficking of wild-type and variant (ΔF508) cystic fibrosis transmembrane conductance regulator (CFTR) responsible for the inherited disease cystic fibrosis (CF). We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client. We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.