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.     

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 Hsp90 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.     

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.     

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.     

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.     

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.     

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.     

Plasticity of the Hsp90 chaperone machine in divergent eukaryotic organisms

Hsp90 is critical for the regulation and activation of numerous client proteins critical for diverse functions such as cell growth, differentiation, and reproduction. Cytosolic Hsp90 function is dependent on a battery of co-chaperone proteins that regulate the ATPase activity of Hsp90 function or direct Hsp90 to interact with specific client proteins. Little is known about how Hsp90 complexes vary between different organisms and how this affects the scope of clients that are activated by Hsp90. This study determined whether ten distinct Hsp90 co-chaperones were encoded by genes in 19 disparate eukaryotic organisms. Surprisingly, none of the co-chaperones were present in all organisms. The co-chaperone Hop/Sti1 was most widely dispersed (18 out of 19 species), while orthologs of Cdc37, which is critical for the stability and activation of diverse protein kinases in yeast and mammals, were identified in only nine out of 19 species examined. The organism with the smallest proteome, Encephalitozoon cuniculi, contained only three of these co-chaperones, suggesting a correlation between client diversity and the complexity of the Hsp90 co-chaperone machine. Our results suggest co-chaperones are critical for cytosolic Hsp90 function in vivo, but that the composition of Hsp90 complexes varies depending on the specialized protein folding requirements of divergent species.     

A review of multi-domain and flexible molecular chaperones studies by small-angle X-ray scattering

Intrinsic flexibility is closely related to protein function, and a plethora of important regulatory proteins have been found to be flexible, multi-domain or even intrinsically disordered. On the one hand, understanding such systems depends on how these proteins behave in solution. On the other, small-angle X-ray scattering (SAXS) is a technique that fulfills the requirements to study protein structure and dynamics relatively quickly with few experimental limitations. Molecular chaperones from Hsp70 and Hsp90 families are multi-domain proteins containing flexible and/or disordered regions that play central roles in cellular proteostasis. Here, we review the structure and function of these proteins by SAXS. Our general approach includes the use of SAXS data to determine size and shape parameters, as well as protein shape reconstruction and their validation by using accessory biophysical tools. Some remarkable examples are presented that exemplify the potential of the SAXS technique. Protein structure can be determined in solution even at limiting protein concentrations (for example, human mortalin, a mitochondrial Hsp70 chaperone). The protein organization, flexibility and function (for example, the J-protein co-chaperones), oligomeric status, domain organization, and flexibility (for the Hsp90 chaperone and the Hip and Hep1 co-chaperones) may also be determined. Lastly, the shape, structural conservation, and protein dynamics (for the Hsp90 chaperone and both p23 and Aha1 co-chaperones) may be studied by SAXS. We believe this review will enhance the application of the SAXS technique to the study of the molecular chaperones.     

Hsp90 structure and function studied by NMR spectroscopy

The molecular chaperone Hsp90 plays a crucial role in folding and maturation of regulatory proteins. Key aspects of Hsp90's molecular mechanism and its adenosine-5'-triphosphate (ATP)-controlled active cycle remain elusive. In particular the role of conformational changes during the ATPase cycle and the molecular basis of the interactions with substrate proteins are poorly understood. The dynamic nature of the Hsp90 machine designates nuclear magnetic resonance (NMR) spectroscopy as an attractive method to unravel both the chaperoning mechanism and interaction with partner proteins. NMR is particularly suitable to provide a dynamic picture of protein-protein interactions at atomic resolution. Hsp90 is rather a challenging protein for NMR studies, due to its high molecular weight and its structural flexibility. The recent technologic advances allowed overcoming many of the traditional obstacles. Here, we describe the different approaches that allowed the investigation of Hsp90 using state-of-the-art NMR methods and the results that were obtained. NMR spectroscopy contributed to understanding Hsp90's interaction with the co-chaperones p23, Aha1 and Cdc37. A particular exciting prospect of NMR, however, is the analysis of Hsp90 interaction with substrate proteins. Here, the ability of this method to contribute to the structural characterization of not fully folded proteins becomes crucial. Especially the interaction of Hsp90 with one of its natural clients, the tumour suppressor p53, has been intensively studied by NMR spectroscopy. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

Contribution of N- and C-terminal domains to the function of Hsp90 in Saccharomyces cerevisiae

The molecular chaperone Hsp90 is a regulatory component of some key signalling proteins in the cytosol of eukaryotic cells. For some of these functions, its interaction with co-chaperones is required. Limited proteolysis defined stable folded units of Hsp90. Both an N-terminal (N210) and a C-terminal (262C) fragment interact with non-native substrate proteins in vitro, but with different specificity and ATP dependence. Here, we analysed the functional properties of these Hsp90 fragments in vivo and in vitro. We determined their influence on the general viability and cell growth of Saccharomyces cerevisiae. Expression of N210 or 262C resulted in a dominant-negative phenotype in several yeast strains tested. Their expression was not toxic, but inhibited cell growth. Further, both were unable to restore viability to Hsp90-depleted cells. In addition, N210 and 262C influence the maturation of Hsp90 substrates, such as the glucocorticoid receptor and pp60v-Src kinase. Specifically, 262C forms partially active chaperone complexes, leading to an arrest of the chaperoned substrate at a certain stage of its maturation cycle. This demonstrates the requirement of a sophisticated and cofactor-regulated interplay between N- and C-terminal activities for Hsp90 function in vivo.     

Molecular analysis of Plasmodium falciparum co-chaperone Aha1 supports its interaction with and regulation of Hsp90 in the malaria parasite

The recent recognition of Plasmodium falciparum Hsp90 (PfHsp90) as a promising anti-malaria drug target has sparked interest in identifying factors that regulate its function and drug-interaction. Co-chaperones are well-known regulators of Hsp90's chaperone function, and certain members have been implicated in conferring protection against lethal cellular effects of Hsp90-specific inhibitors. In this context, studies on PfHsp90's co-chaperones are imperative to gain insight into the regulation of the chaperone in the malaria parasite. In this study, a putative co-chaperone P. falciparum Aha1 (PfAha1) was identified and investigated for its interaction and regulation of PfHsp90. A previous genome-wide yeast two-hybrid study failed to identify PfAha1's association with PfHsp90, which prompted us to use a directed assay to investigate their interaction. PfAha1 was shown to interact with PfHsp90 via the in vivo split-ubiquitin assay and the association was confirmed in vitro by GST pull-down experiments. The GST pull-down assay further revealed PfAha1's interaction with PfHsp90 to be dependent on MgCl₂ and ATP, and was competed by co-chaperone Pfp23 that binds PfHsp90 under the same condition. In addition, the PfHsp90-PfAha1 complex was found to be sensitive to disruption by high salt, indicating a polar interaction between them. Using bio-computational modelling coupled with site-directed mutagenesis, the polar residue N108 in PfAha1 was found to be strategically located and essential for PfHsp90 interaction. The functional significance of PfAha1's interaction was clearly that of exerting a stimulatory effect on the ATPase activity of PfHsp90, likely to be essential for promoting the activation of PfHsp90's client proteins.     

The heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized ATP-binding domain in the carboxyl terminus of the chaperone

Heat shock protein 90 (Hsp90), one of the most abundant chaperones in eukaryotes, participates in folding and stabilization of signal-transducing molecules including steroid hormone receptors and protein kinases. The amino terminus of Hsp90 contains a non-conventional nucleotide-binding site, related to the ATP-binding motif of bacterial DNA gyrase. The anti-tumor agents geldanamycin and radicicol bind specifically at this site and induce destabilization of Hsp90-dependent client proteins. We recently demonstrated that the gyrase inhibitor novobiocin also interacts with Hsp90, altering the affinity of the chaperone for geldanamycin and radicicol and causing in vitro and in vivo depletion of key regulatory Hsp90-dependent kinases including v-Src, Raf-1, and p185(ErbB2). In the present study we used deletion/mutation analysis to identify the site of interaction of novobiocin with Hsp90, and we demonstrate that the novobiocin-binding site resides in the carboxyl terminus of the chaperone. Surprisingly, this motif also recognizes ATP, and ATP and novobiocin efficiently compete with each other for binding to this region of Hsp90. Novobiocin interferes with association of the co-chaperones Hsc70 and p23 with Hsp90. These results identify a second site on Hsp90 where the binding of small molecule inhibitors can significantly impact the function of this chaperone, and they support the hypothesis that both amino- and carboxyl-terminal domains of Hsp90 interact to modulate chaperone activity.     

Functional interactions between Hsp90 and the co-chaperones Cns1 and Cpr7 in Saccharomyces cerevisiae

Hsp90 complexes contain a class of co-chaperones characterized by a tetratricopeptide repeat (TPR) domain, which mediates binding to a carboxyl-terminal EEVD region in Hsp90. Among Hsp90 TPR co-chaperones in Saccharomyces cerevisiae, only Cns1 is essential. The amino terminus of Cns1, which harbors the TPR domain, is sufficient for viability when overexpressed. In a screen for temperature-sensitive alleles of CNS1, we identified mutations resulting in substitutions of conserved residues in the TPR domain. Mutations in CNS1 disrupt in vitro and in vivo interaction with Hsp90 and reduce Hsp90 function, indicating that Cns1 is a bona fide co-chaperone. Genetic interactions between CNS1 and another Hsp90 co-chaperone, CPR7, suggest that the two co-chaperones share an essential role in the cell. Although both the TPR and the isomerase domains of the cyclophilin Cpr7 are required for viability of cns1 mutant cells, this requirement does not depend on the catalytic function of the isomerase domain. Instead, hydrophilic residues on the surface of this domain appear to be important for the common Cns1.Cpr7 function. Although both co-chaperones interact with Hsp90 primarily through the carboxyl terminus (EEVD), Cns1 and Cpr7 are mostly found in complexes distinct from Hsp90. EEVD is required for normal growth in cns1 mutant cells, demonstrating for the first time in vivo requirement for this conserved region of Hsp90. Overall, our findings reveal a considerable degree of complexity in the interactions not only between Hsp90 and its co-chaperones, but also among the co-chaperones themselves.     

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.     

Thr90 phosphorylation of Hsp90α by protein kinase A regulates its chaperone machinery

Hsp90 (heat-shock protein 90) is one of the most important molecular chaperones in eukaryotes. Hsp90 facilitates the maturation, activation or degradation of its client proteins. It is now well accepted that both ATP binding and co-chaperone association are involved in regulating the Hsp90 chaperone machinery. However, other factors such as post-translational modifications are becoming increasingly recognized as being involved in this process. Recent studies have reported that phosphorylation of Hsp90 plays an unanticipated role in this process. In the present study, we systematically investigated the impact of phosphorylation of a single residue (Thr90) of Hsp90α (pThr90-Hsp90α) on its chaperone machinery. We demonstrate that protein kinase A specifically phosphorylates Hsp90α at Thr90, and that the pThr9090-Hsp90α level is significantly elevated in proliferating cells. Thr90 phosphorylation affects the binding affinity of Hsp90α to ATP. Subsequent examination of the interactions of Hsp90α with co-chaperones reveals that Thr90 phosphorylation specifically regulates the association of a subset of co-chaperones with Hsp90α. The Hsp90α T90E phosphor-mimic mutant exhibits increased association with Aha1 (activator of Hsp90 ATPase homologue 1), p23, PP5 (protein phosphatase 5) and CHIP (C-terminus of Hsp70-interacting protein), and decreased binding affinity with Hsp70, Cdc37 (cell division cycle 37) and Hop [Hsc70 (heat-shock cognate protein 70)/Hsp90-organizing protein], whereas its interaction with FKBP52 (FK506-binding protein 4) is only moderately affected. Moreover, we find that the ability of the T90E mutant to form complexes with its clients, such as Src, Akt or PKCγ (protein kinase Cγ), is dramatically impaired, suggesting that phosphorylation affects its chaperoning activity. Taken together, the results of the present study demonstrate that Thr90 phosphorylation is actively engaged in the regulation of the Hsp90α chaperone machinery and should be a generic determinant for the cycling of Hsp90α chaperone function.     

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.     

Interaction of Heat Shock Protein 90 and the Co-chaperone Cpr6 with Ura2, a Bifunctional Enzyme Required for Pyrimidine Biosynthesis

The molecular chaperone heat shock protein 90 (Hsp90) is an essential protein required for the activity and stability of multiple proteins termed clients. Hsp90 cooperates with a set of co-chaperone proteins that modulate Hsp90 activity and/or target clients to Hsp90 for folding. Many of the Hsp90 co-chaperones, including Cpr6 and Cpr7, contain tetratricopeptide repeat (TPR) domains that bind a common acceptor site at the carboxyl terminus of Hsp90. We found that Cpr6 and Hsp90 interacted with Ura2, a protein critical for pyrimidine biosynthesis. Mutation or inhibition of Hsp90 resulted in decreased accumulation of Ura2, indicating it is an Hsp90 client. Cpr6 interacted with Ura2 in the absence of stable Cpr6-Hsp90 interaction, suggesting a direct interaction. However, loss of Cpr6 did not alter the Ura2-Hsp90 interaction or Ura2 accumulation. The TPR domain of Cpr6 was required for Ura2 interaction, but other TPR containing co-chaperones, including Cpr7, failed to interact with Ura2 or rescue CPR6-dependent growth defects. Further analysis suggests that the carboxyl-terminal 100 amino acids of Cpr6 and Cpr7 are critical for specifying their unique functions, providing new information about this important class of Hsp90 co-chaperones.     

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.     

The 'active life' of Hsp90 complexes

Hsp90 forms a variety of complexes differing both in clientele and co-chaperones. Central to the role of co-chaperones in the formation of Hsp90 complexes is the delivery of client proteins and the regulation of the ATPase activity of Hsp90. Determining the mechanisms by which co-chaperones regulate Hsp90 is essential in understanding the assembly of these complexes and the activation and maturation of Hsp90's clientele. Mechanistically, co-chaperones alter the kinetics of the ATP-coupled conformational changes of Hsp90. The structural changes leading to the formation of a catalytically active unit involve all regions of the Hsp90 dimer. Their complexity has allowed different orthologues of Hsp90 to evolve kinetically in slightly different ways. The interaction of the cytosolic Hsp90 with a variety of co-chaperones lends itself to a complex set of different regulatory mechanisms that modulate Hsp90's conformation and ATPase activity. It also appears that the conformational switches of Hsp90 are not necessarily coupled under all circumstances. Here, I described different co-chaperone complexes and then discuss in detail the mechanisms and role that specific co-chaperones play in this. I will also discuss emerging evidence that post-translational modifications also affect the ATPase activity of Hsp90, and thus complex formation. Finally, I will present evidence showing how Hsp90's active site, although being highly conserved, can be altered to show resistance to drug binding, but still maintain ATP binding and ATPase activity. Such changes are therefore unlikely to significantly alter Hsp90's interactions with client proteins and co-chaperones. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).