Processing of proteins by the molecular chaperone Hsp104

The molecular chaperone Hsp104 is an AAA+ ATPase (ATPase associated with a variety of cellular activities) from yeast that catalyzes protein disaggregation. Using mutagenesis, we impaired nucleotide binding or hydrolysis in the two nucleotide-binding domains (NBD) of Hsp104 and analyzed the consequences for chaperone function by monitoring ATP hydrolysis, polypeptide binding, polypeptide processing, and disaggregation. Our results reveal that ATP binding to NBD1 serves as a central regulatory switch for the chaperone; it triggers binding of polypeptides, and stimulates ATP hydrolysis in the C-terminal NBD2 by more than two orders of magnitude, implying that ATP hydrolysis in this domain is important for disaggregation. Moreover, we show that Hsp104 actively unfolds its polypeptide substrates during processing, demonstrating that AAA+ proteins involved in disaggregation share a common threading mechanism with AAA+ proteins mediating protein unfolding/degradation.     

Thermal adaptation of the yeast mitochondrial Hsp70 system is regulated by the reversible unfolding of its nucleotide exchange factor

The Hsp70 protein switches during its functional cycle from an ADP-bound state with a high affinity for substrates to a low-affinity, ATP-bound state, with concomitant release of the client protein. The rate of the chaperone cycle is regulated by co-chaperones such as nucleotide exchange factors that significantly accelerate the ADP/ATP exchange. Mge1p, a mitochondrial matrix protein with homology to bacterial GrpE, serves as the nucleotide exchange factor of mitochondrial Hsp70. Here, we analyze the influence of temperature on the structure and functional properties of Mge1p from the yeast Saccharomyces cerevisiae. Mge1p is a dimer in solution that undergoes a reversible thermal transition at heat-shock temperatures, i.e. above 37 degrees C, that involves protein unfolding and dimer dissociation. The thermally denatured protein is unable to interact stably with mitochondrial Hsp70, and therefore is unable to regulate its ATPase and chaperone cycle. Crosslinking of wild-type mitochondria reveals that Mge1p undergoes the same dimer to monomer temperature-dependent shift, and that the nucleotide exchange factor does not associate with its Hsp70 partner at stress temperatures (i.e. > or =45 degrees C). Once the stress conditions disappear, Mge1p refolds and recovers both structure and functional properties. Therefore, Mge1p can act as a thermosensor for the mitochondrial Hsp70 system, regulating the nucleotide exchange rates under heat shock, as has been described for two bacterial GrpE proteins. The thermosensor activity is conserved in the GrpE-like nucleotide exchange factors although, as discussed here, it is achieved through a different structural mechanism.     

Stimulation of the weak ATPase activity of human hsp90 by a client protein.

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in the folding and assembly of a limited set of "client" proteins, many of which are involved in signal transduction pathways. In vivo, it is found in complex with additional proteins, including the chaperones Hsp70, Hsp40, Hip and Hop (Hsp-interacting and Hsp-organising proteins, respectively), as well as high molecular mass immunophilins, such as FKBP59, and the small acidic protein p23. The role of these proteins in Hsp90-mediated assembly processes is poorly understood. It is known that ATP binding and hydrolysis are essential for Hsp90 function in vivo and in vitro.Here we show, for the first time, that human Hsp90 has ATPase activity in vitro. The ATPase activity is characterised using a sensitive assay based on a chemically modified form of the phosphate-binding protein from Escherichia coli. Human Hsp90 is a very weak ATPase, its activity is significantly lower than that of the yeast homologue, and it has a half-life of ATP hydrolysis of eight minutes at 37 degrees C. Using a physiological substrate of Hsp90, the ligand-binding domain of the glucocorticoid receptor, we show that this "client" protein can stimulate the ATPase activity up to 200-fold. This effect is highly specific and unfolded or partially folded proteins, which are known to bind to Hsp90, do not affect the ATPase activity. In addition, the peroxisome proliferator-activated receptor, which is related in both sequence and structure to the glucocorticoid receptor but which does not bind Hsp90, has no observable effect on the ATPase activity.We establish the effect of the co-chaperones Hop, FKBP59 and p23 on the basal ATPase activity as well as the client protein-stimulated ATPase activity of human Hsp90. In contrast with the yeast system, human Hop has little effect on the basal rate of ATP hydrolysis but significantly inhibits the client-protein stimulated rate. Similarly, FKBP59 has little effect on the basal rate but stimulates the client-protein stimulated rate further. In contrast, p23 inhibits both the basal and stimulated rates of ATP hydrolysis.Our results show that the ATPase activity of human Hsp90 is highly regulated by both client protein and co-chaperone binding. We suggest that the rate of ATP hydrolysis is critical to the mode of action of Hsp90, consistent with results that have shown that both over and under-active ATPase mutants of yeast Hsp90 have impaired function in vivo. We suggest that the tight regulation of the ATPase activity of Hsp90 is important and allows the client protein to remain bound to Hsp90 for sufficient time for activation to occur.     

The prion curing agent guanidinium chloride specifically inhibits ATP hydrolysis by Hsp104

The molecular chaperone Hsp104 from Saccharomyces cerevisiae dissolves protein aggregates in the cell and is thus of crucial importance for the thermotolerance of yeast. In addition to this disaggregase activity, Hsp104 has a key function in yeast prion propagation, as Hsp104 was found to be essential for the maintenance of the associated phenotypes. In vivo data suggest that Hsp104 function is affected by guanidinium chloride. Adding small amounts of this compound to yeast medium causes curing of the prions: cells lose their prion-related phenotype. Guanidinium chloride was also found to impair heat shock resistance. Here, we present a detailed in vitro analysis showing that guanidinium chloride is an uncompetitive inhibitor of Hsp104. Micromolar concentrations of this agent reduce the ATPase activity of Hsp104 to approximately 35% of its normal activity. This inhibition is not related to the denaturing properties of this compound, because Hsp104 was not affected by urea. Guanidinium ions selectively bind to the nucleotide-bound, hexameric state of the molecular chaperone. Thus, they increase the affinity of Hsp104 for adenine nucleotides and promote the nucleotide-dependent oligomerization of the chaperone. Our findings strongly suggest that guanidinium chloride causes curing of yeast prions by perturbing the ATPase of Hsp104, which is essential for both prion propagation and thermotolerance.     

ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo.

Hsp90 is an abundant molecular chaperone essential to the establishment of many cellular regulation and signal transduction systems, but remains one of the least well described chaperones. The biochemical mechanism of protein folding by Hsp90 is poorly understood, and the direct involvement of ATP has been particularly contentious. Here we demonstrate in vitro an inherent ATPase activity in both yeast Hsp90 and the Escherichia coli homologue HtpG, which is sensitive to inhibition by the Hsp90-specific antibiotic geldanamycin. Mutations of residues implicated in ATP binding and hydrolysis by structural studies abolish this ATPase activity in vitro and disrupt Hsp90 function in vivo. These results show that Hsp90 is directly ATP dependent in vivo, and suggest an ATP-coupled chaperone cycle for Hsp90-mediated protein folding.     

Cer1p Functions as a Molecular Chaperone in the Endoplasmic Reticulum of Saccharomyces cerevisiae

Cer1p/Lhs1p/Ssi1p is a novel Hsp70-related protein that is important for the translocation of a subset of proteins into the yeast Saccharomyces cerevisiae endoplasmic reticulum. Cer1p has very limited amino acid identity to the hsp70 chaperone family in the N-terminal ATPase domain but lacks homology to the highly conserved hsp70 peptide binding domain. The role of Cer1p in protein folding and translocation was assessed. Deletion of CER1 slowed the folding of reduced pro-carboxypeptidase Y (pro-CPY) approximately twofold in yeast. In wild-type yeast under reducing conditions, pro-CPY can be found in a complex with Cer1p, while partially purified Cer1p is able to bind directly to peptides. Together, this suggests that Cer1p has a chaperoning activity required for proper refolding of denatured pro-CPY which is mediated by direct interaction with the unfolded polypeptide. Cer1p peptide binding and oligomerization could be disrupted by addition of ATP, confirming that Cer1p possesses a functional ATP binding site, much like Kar2p and other members of the hsp70 family. Interestingly, replacing the signal sequence of a CER1-dependent protein with that of a CER1-independent protein did not relieve the requirement of CER1 for import. This result suggests that an interaction with the mature portion of the protein also is important for the translocation role of Cer1p. The CER1 RNA levels increase at lower temperatures. In addition, the effects of deletion on folding and translocation are more severe at lower temperatures. Therefore, these results suggest that Cer1p provides an additional chaperoning activity in processes known to require Kar2p. However, there appears to be a greater requirement for Cer1p chaperone activity at lower temperatures.     

The disaggregation activity of the mitochondrial ClpB homolog Hsp78 maintains Hsp70 function during heat stress

Molecular chaperones are important components of mitochondrial protein biogenesis and are required to maintain the organellar function under normal and stress conditions. We addressed the functional role of the Hsp100/ClpB homolog Hsp78 during aggregation reactions and its functional cooperation with the main mitochondrial Hsp70, Ssc1, in mitochondria of the yeast Saccharomyces cerevisiae. By establishing an aggregation/disaggregation assay in intact mitochondria we demonstrated that Hsp78 is indispensable for the resolubilization of protein aggregates generated by heat stress under in vivo conditions. The ATP-dependent disaggregation activity of Hsp78 was capable of reversing the preprotein import defect of a destabilized mutant form of Ssc1. This role in disaggregation of Ssc1 is unique for Hsp78, since the recently identified, Hsp70-specific chaperone Zim17 had no effect on the resolubilization reaction. We observed only a minor effect of the second mitochondrial Hsp100 family member Mcx1 on protein disaggregation. A "holding" activity of the mitochondrial Hsp70 system was a prerequisite for a successful resolubilization of aggregated proteins. We conclude that the protective role of Hsp78 in thermotolerance is mainly based on maintaining the molecular chaperone Ssc1 in a soluble and functional state.     

Roles of individual domains and conserved motifs of the AAA+ chaperone ClpB in oligomerization,ATP hydrolysis,and chaperone activity

ClpB of Escherichia coli is an ATP-dependent ring-forming chaperone that mediates the resolubilization of aggregated proteins in cooperation with the DnaK chaperone system. ClpB belongs to the Hsp100/Clp subfamily of AAA+ proteins and is composed of an N-terminal domain and two AAA-domains that are separated by a "linker" region. Here we present a detailed structure-function analysis of ClpB, dissecting the individual roles of ClpB domains and conserved motifs in oligomerization, ATP hydrolysis, and chaperone activity. Our results show that ClpB oligomerization is strictly dependent on the presence of the C-terminal domain of the second AAA-domain, while ATP binding to the first AAA-domains stabilized the ClpB oligomer. Analysis of mutants of conserved residues in Walker A and B and sensor 2 motifs revealed that both AAA-domains contribute to the basal ATPase activity of ClpB and communicate in a complex manner. Chaperone activity strictly depends on ClpB oligomerization and the presence of a residual ATPase activity. The N-domain is dispensable for oligomerization and for the disaggregating activity in vitro and in vivo. In contrast the presence of the linker region, although not involved in oligomerization, is essential for ClpB chaperone activity.     

Coupling and dynamics of subunits in the hexameric AAA+ chaperone ClpB

The bacterial AAA+ protein ClpB and its eukaryotic homologue Hsp104 ensure thermotolerance of their respective organisms by reactivating aggregated proteins in cooperation with the Hsp70/Hsp40 chaperone system. Like many members of the AAA+ superfamily, the ClpB protomers form ringlike homohexameric complexes. The mechanical energy necessary to disentangle protein aggregates is provided by ATP hydrolysis at the two nucleotide-binding domains of each monomer. Previous studies on ClpB and Hsp104 show a complex interplay of domains and subunits resulting in homotypic and heterotypic cooperativity. Using mutations in the Walker A and Walker B nucleotide-binding motifs in combination with mixing experiments we investigated the degree of inter-subunit coupling with respect to different aspects of the ClpB working cycle. We find that subunits are tightly coupled with regard to ATPase and chaperone activity, but no coupling can be observed for ADP binding. Comparison of the data with statistical calculations suggests that for double Walker mutants, approximately two in six subunits are sufficient to abolish chaperone and ATPase activity completely. In further experiments, we determined the dynamics of subunit reshuffling. Our results show that ClpB forms a very dynamic complex, reshuffling subunits on a timescale comparable to steady-state ATP hydrolysis. We propose that this could be a protection mechanism to prevent very stable aggregates from becoming suicide inhibitors for ClpB.     

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.     

In Vivo Function of Hsp90 Is Dependent on ATP Binding and ATP Hydrolysis

Heat shock protein 90 (Hsp90), an abundant molecular chaperone in the eukaryotic cytosol, is involved in the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. The basic mechanism of action of Hsp90 is not yet understood. In particular, it has been debated whether Hsp90 function is ATP dependent. A recent crystal structure of the NH₂-terminal domain of yeast Hsp90 established the presence of a conserved nucleotide binding site that is identical with the binding site of geldanamycin, a specific inhibitor of Hsp90. The functional significance of nucleotide binding by Hsp90 has remained unclear. Here we present evidence for a slow but clearly detectable ATPase activity in purified Hsp90. Based on a new crystal structure of the NH₂-terminal domain of human Hsp90 with bound ADP-Mg and on the structural homology of this domain with the ATPase domain of Escherichia coli DNA gyrase, the residues of Hsp90 critical in ATP binding (D93) and ATP hydrolysis (E47) were identified. The corresponding mutations were made in the yeast Hsp90 homologue, Hsp82, and tested for their ability to functionally replace wild-type Hsp82. Our results show that both ATP binding and hydrolysis are required for Hsp82 function in vivo. The mutant Hsp90 proteins tested are defective in the binding and ATP hydrolysis–dependent cycling of the co-chaperone p23, which is thought to regulate the binding and release of substrate polypeptide from Hsp90. Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intricate allosteric communication between the domains of the Hsp90 dimer. Our results establish Hsp90 as an ATP-dependent chaperone.     

Conserved conformational changes in the ATPase cycle of human Hsp90

The dimeric molecular chaperone Hsp90 is required for the activation and stabilization of hundreds of substrate proteins, many of which participate in signal transduction pathways. The activation process depends on the hydrolysis of ATP by Hsp90. Hsp90 consists of a C-terminal dimerization domain, a middle domain, which may interact with substrate protein, and an N-terminal ATP-binding domain. A complex cycle of conformational changes has been proposed for the ATPase cycle of yeast Hsp90, where a critical step during the reaction requires the transient N-terminal dimerization of the two protomers. The ATPase cycle of human Hsp90 is less well understood, and significant differences have been proposed regarding key mechanistic aspects. ATP hydrolysis by human Hsp90alpha and Hsp90beta is 10-fold slower than that of yeast Hsp90. Despite these differences, our experiments suggest that the underlying enzymatic mechanisms are highly similar. In both cases, a concerted conformational rearrangement involving the N-terminal domains of both subunits is controlling the rate of ATP turnover, and N-terminal cross-talk determines the rate-limiting steps. Furthermore, similar to yeast Hsp90, the slow ATP hydrolysis by human Hsp90s can be stimulated up to over 100-fold by the addition of the co-chaperone Aha1 from either human or yeast origin. Together, our results show that the basic principles of the Hsp90 ATPase reaction are conserved between yeast and humans, including the dimerization of the N-terminal domains and its regulation by the repositioning of the ATP lid from its original position to a catalytically competent one.     

Characterization of the Hsp100 disaggregase from sugarcane (SHsp101) for chaperone like activity in a yeast system

The Hsp104/ClpB protein subfamily is unique in its ability to reactivate protein aggregates, which are implicated in several human and animal diseases. However, when compared to the unicellular yeast Hsp104 and bacterial ClpB disaggregases, the multicellular plant ortholog (Hsp101) is poorly studied. Here, we present a functional characterization of the Hsp101 (SHsp101) from the economically and agriculturally important sugarcane cultivar. Like Hsp104, SHsp101 has in vitro ATP/ATPγS dependent aggregate reactivation activity, confers induced thermotolerance in yeast and its ATPase activity is sensitive to guanidinium chloride. However, SHsp101 has distinct properties not previously reported for Hsp104, including higher ATPase activity at elevated temperatures and ATP independent intrinsic chaperone activity. Hence, SHsp101 has overlapping and distinct features from Hsp104.     

Hsp78 chaperone functions in restoration of mitochondrial network following heat stress

Under physiological conditions mitochondria of yeast Saccharomyces cerevisiae form a branched tubular network, the continuity of which is maintained by balanced membrane fusion and fission processes. Here, we show using mitochondrial matrix targeted green fluorescent protein that exposure of cells to extreme heat shock led to dramatic changes in mitochondrial morphology, as tubular network disintegrated into several fragmented vesicles. Interestingly, this fragmentation did not affect mitochondrial ability to maintain the membrane potential. Cells subjected to recovery at physiological temperature were able to restore the mitochondrial network, as long as an active matrix chaperone, Hsp78, was present. Deletion of HSP78 gene did not affect fragmentation of mitochondria upon heat stress, but significantly inhibited ability to restore mitochondrial network. Changes of mitochondrial morphology correlated with aggregation of mitochondrial proteins. On the other hand, recovery of mitochondrial network correlated with disappearance of protein aggregates and reactivation of enzymatic activity of a model thermo-sensitive protein: mitochondrial DNA polymerase. Since protein disaggregation and refolding is mediated by Hsp78 chaperone collaborating with Hsp70 chaperone system, we postulate that effect of Hsp78 on mitochondrial morphology upon recovery after heat shock is mediated by its ability to restore activity of unknown protein(s) responsible for maintenance of mitochondrial morphology.     

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.     

The metalloprotease encoded by ATP23 has a dual function in processing and assembly of subunit 6 of mitochondrial ATPase

In the present study we have identified a new metalloprotease encoded by the nuclear ATP23 gene of Saccharomyces cerevisiae that is essential for expression of mitochondrial ATPase (F(1)-F(O) complex). Mutations in ATP23 cause the accumulation of the precursor form of subunit 6 and prevent assembly of F(O). Atp23p is associated with the mitochondrial inner membrane and is conserved from yeast to humans. A mutant harboring proteolytically inactive Atp23p accumulates the subunit 6 precursor but is nonetheless able to assemble a functional ATPase complex. These results indicate that removal of the subunit 6 presequence is not an essential event for ATPase biogenesis and that Atp23p, in addition to its processing activity, must provide another important function in F(O) assembly. The product of the yeast ATP10 gene was previously shown to interact with subunit 6 and to be required for its association with the subunit 9 ring. In this study one extra copy of ATP23 was found to be an effective suppressor of an atp10 null mutant, suggesting an overlap in the functions of Atp23p and Atp10p. Atp23p may, therefore, also be a chaperone, which in conjunction with Atp10p mediates the association of subunit 6 with the subunit 9 ring.     

Coordinated ATP hydrolysis by the Hsp90 dimer.

The Hsp90 dimer is a molecular chaperone with an unusual N-terminal ATP binding site. The structure of the ATP binding site makes it a member of a new class of ATP-hydrolyzing enzymes, known as the GHKL family. While for some of the family members structural data on conformational changes occurring after ATP binding are available, these are still lacking for Hsp90. Here we set out to investigate the correlation between dimerization and ATP hydrolysis by Hsp90. The dimerization constant of wild type (WT) Hsp90 was determined to be 60 nm. Heterodimers of WT Hsp90 with fragments lacking the ATP binding domain form readily and exhibit dimerization constants similar to full-length Hsp90. However, the ATPase activity of these heterodimers was significantly lower than that of the wild type protein, indicating cooperative interactions in the N-terminal part of the protein that lead to the activation of the ATPase activity. To further address the contribution of the N-terminal domains to the ATPase activity, we used an Hsp90 point mutant that is unable to bind ATP. Since heterodimers between the WT protein and this mutant showed WT ATPase activity, this mutant, although unable to bind ATP, still has the ability to stimulate the activity in its WT partner domain. Thus, contact formation between the N-terminal domains might not depend on ATP bound to both domains. Together, these results suggest a mechanism for coupling the hydrolysis of ATP to the opening-closing movement of the Hsp90 molecular chaperone.     

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.