Regulation of Hsp70 function by HspBP1: structural analysis reveals an alternate mechanism for Hsp70 nucleotide exchange

HspBP1 belongs to a family of eukaryotic proteins recently identified as nucleotide exchange factors for Hsp70. We show that the S. cerevisiae ortholog of HspBP1, Fes1p, is required for efficient protein folding in the cytosol at 37 degrees C. The crystal structure of HspBP1, alone and complexed with part of the Hsp70 ATPase domain, reveals a mechanism for its function distinct from that of BAG-1 or GrpE, previously characterized nucleotide exchange factors of Hsp70. HspBP1 has a curved, all alpha-helical fold containing four armadillo-like repeats unlike the other nucleotide exchange factors. The concave face of HspBP1 embraces lobe II of the ATPase domain, and a steric conflict displaces lobe I, reducing the affinity for nucleotide. In contrast, BAG-1 and GrpE trigger a conserved conformational change in lobe II of the ATPase domain. Thus, nucleotide exchange on eukaryotic Hsp70 occurs through two distinct mechanisms.     

Domain:domain interactions within Hop, the Hsp70/Hsp90 organizing protein, are required for protein stability and structure

The major heat shock protein (Hsp) chaperones Hsp70 and Hsp90 both bind the co-chaperone Hop (Hsp70/Hsp90 organizing protein), which coordinates Hsp actions in folding protein substrates. Hop contains three tetratricopeptide repeat (TPR) domains that have binding sites for the conserved EEVD C termini of Hsp70 and Hsp90. Crystallographic studies have shown that EEVD interacts with positively charged amino acids in Hop TPR-binding pockets (called carboxylate clamps), and point mutations of these carboxylate clamp positions can disrupt Hsp binding. In this report, we use circular dichroism to assess the effects of point mutations and Hsp70/Hsp90 peptide binding on Hop conformation. Our results show that Hop global conformation is destabilized by single point mutations in carboxylate clamp positions at pH 5, while the structure of individual TPR domains is unaffected. Binding of peptides corresponding to the C termini of Hsp70 and Hsp90 alters the global conformation of wild-type Hop, whereas peptide binding does not alter conformation of individual TPR domains. These results provide biophysical evidence that Hop-binding pockets are directly involved with domain:domain interactions, both influencing Hop global conformation and Hsp binding, and contributing to proper coordination of Hsp70 and Hsp90 interactions with protein substrates.     

Hsp70 Cochaperones HspBP1 and BAG-1M Differentially Regulate Steroid Hormone Receptor Function

Hsp70 binding protein 1 (HspBP1) and Bcl2-associated athanogene 1 (BAG-1), the functional orthologous nucleotide exchange factors of the heat shock protein 70 kilodalton (Hsc70/Hsp70) chaperones, catalyze the release of ADP from Hsp70 while inducing different conformational changes of the ATPase domain of Hsp70. An appropriate exchange rate of ADP/ATP is crucial for chaperone-dependent protein folding processes. Among Hsp70 client proteins are steroid receptors such as the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), and the androgen receptor (AR). BAG-1 diversely affects steroid receptor activity, while to date the influence of HspBP1 on steroid receptor function is mostly unknown. Here, we compared the influence of HspBP1 and BAG-1M on Hsp70-mediated steroid receptor folding complexes and steroid receptor activity. Coimmunoprecipitation studies indicated preferential binding of Hsp40 and the steroid receptors to BAG-1M as compared to HspBP1. Furthermore, Hsp70 binding to the ligand-binding domain of GR was reduced in the presence of HspBP1 but not in the presence of BAG-1M as shown by pull-down assays. Reporter gene experiments revealed an inhibitory effect on GR, MR, and AR at a wide range of HspBP1 protein levels and at hormone concentrations at or approaching saturation. BAG-1M exhibited a transition from stimulatory effects at low BAG-1M levels to inhibitory effects at higher BAG-1M levels. Overall, BAG-1M and HspBP1 had differential impacts on the dynamic composition of steroid receptor folding complexes and on receptor function with important implications for steroid receptor physiology.     

Expression of the cochaperone HspBP1 is not coordinately regulated with Hsp70 expression

Intracellular levels of the heat stress protein Hsp70 are elevated following exposure to elevated temperature. The cochaperone HspBP1 is an intracellular protein that is known to bind to and regulate Hsp70 activity. The purpose of this study was to determine if HspBP1 levels changed when Hsp70 levels were altered. Heat stress resulted in an increase in Hsp70 levels but no change in HspBP1 levels. Treatment of cells with the apoptosis inducing drug camptothecin lowered Hsp70 levels but again had no effect on HspBP1 levels. Cells treated with camptothecin plus heat stress did not exhibit an increase in Hsp70 levels. Over-expression in cells stably transfected with HspBP1 cDNA resulted in a 290% increase in HspBP1 levels without a similar change in Hsp70 levels. These results demonstrate that Hsp70 and HspBP1 are not coordinately regulated but provide evidence that an increase in the ratio of HspBP1 to Hsp70 correlates with apoptosis, in a similar way to reducing the amount of Hsp70.     

Anticancer drugs up-regulate HspBP1 and thereby antagonize the prosurvival function of Hsp70 in tumor cells

The 70-kDa heat shock protein (Hsp70) is up-regulated in a wide variety of tumor cell types and contributes to the resistance of these cells to the induction of cell death by anticancer drugs. Hsp70 binding protein 1 (HspBP1) modulates the activity of Hsp70 but its biological significance has remained unclear. We have now examined whether HspBP1 might interfere with the prosurvival function of Hsp70, which is mediated, at least in part, by inhibition of the death-associated permeabilization of lysosomal membranes. HspBP1 was found to be expressed at a higher level than Hsp70 in all normal and tumor cell types examined. Tumor cells with a high HspBP1/Hsp70 molar ratio were more susceptible to anticancer drugs than were those with a low ratio. Ectopic expression of HspBP1 enhanced this effect of anticancer drugs in a manner that was both dependent on the ability of HspBP1 to bind to Hsp70 and sensitive to the induction of Hsp70 by mild heat shock. Furthermore, anticancer drugs up-regulated HspBP1 expression, whereas prevention of such up-regulation by RNA interference reduced the susceptibility of tumor cells to anticancer drugs. Overexpression of HspBP1 promoted the permeabilization of lysosomal membranes, the release of cathepsins from lysosomes into the cytosol, and the activation of caspase-3 induced by anticancer drugs. These results suggest that HspBP1, by antagonizing the prosurvival activity of Hsp70, sensitizes tumor cells to cathepsin-mediated cell death.     

The heat shock-binding protein (HspBP1) protects cells against the cytotoxic action of the Tag7-Hsp70 complex

Heat shock-binding protein HspBP1 is a member of the Hsp70 co-chaperone family. The interaction between HspBP1 and the ATPase domain of the major heat shock protein Hsp70 up-regulates nucleotide exchange and reduces the affinity between Hsp70 and the peptide in its peptide-binding site. Previously we have shown that Tag7 (also known as peptidoglycan recognition protein PGRP-S), an innate immunity protein, interacts with Hsp70 to form a stable Tag7-Hsp70 complex with cytotoxic activity against some tumor cell lines. This complex can be produced in cytotoxic lymphocytes and released during interaction with tumor cells. Here the effect of HspBP1 on the cytotoxic activity of the Tag7-Hsp70 complex was examined. HspBP1 could bind not only to Hsp70, but also to Tag7. This interaction eliminated the cytotoxic activity of Tag7-Hsp70 complex and decreased the ATP concentration required to dissociate Tag7 from the peptide-binding site of Hsp70. Moreover, HspBP1 inhibited the cytotoxic activity of the Tag7-Hsp70 complex secreted by lymphocytes. HspBP1 was detected in cytotoxic CD8+ lymphocytes. This protein was released simultaneously with Tag7-Hsp70 during interaction of these lymphocytes with tumor cells. The simultaneous secretion of the cytotoxic complex with its inhibitor could be a mechanism protecting normal cells from the cytotoxic effect of this complex.     

The crystal structure of the peptide-binding fragment from the yeast Hsp40 protein Sis1

BACKGROUND: Molecular chaperone Hsp40 can bind non-native polypeptide and facilitate Hsp70 in protein refolding. How Hsp40 and other chaperones distinguish between the folded and unfolded states of proteins to bind nonnative polypeptides is a fundamental issue. RESULTS: To investigate this mechanism, we determined the crystal structure of the peptide-binding fragment of Sis1, an essential member of the Hsp40 family from Saccharomyces cerevisiae. The 2.7 A structure reveals that Sis1 forms a homodimer in the crystal by a crystallographic twofold axis. Sis1 monomers are elongated and consist of two domains with similar folds. Sis1 dimerizes through a short C-terminal stretch. The Sis1 dimer has a U-shaped architecture and a large cleft is formed between the two elongated monomers. Domain I in each monomer contains a hydrophobic depression that might be involved in binding the sidechains of hydrophobic amino acids. CONCLUSIONS: Sis1 (1-337), which lacks the dimerization motif, exhibited severe defects in chaperone activity, but could regulate Hsp70 ATPase activity. Thus, dimer formation is critical for Sis1 chaperone function. We propose that the Sis1 cleft functions as a docking site for the Hsp70 peptide-binding domain and that Sis1-Hsp70 interaction serves to facilitate the efficient transfer of peptides from Sis1 to Hsp70.     

Binding of the maize cytosolic Hsp70 to calmodulin, and identification of calmodulin-binding site in Hsp70

Using a gel-overlay technique of biotinylated calmodulin (CaM), we showed that maize cytosolic Hsp70 protein could bind to CaM in the presence of 1 mM CaCl2. The purified maize cytosolic Hsp70 inhibited the activity of CaM-dependent NADK in a concentration-dependent manner. A synthetic peptide, which possesses the 21 amino acid sequence, PRALRRLRTACERAKRTLSST, at positions 261-281 in maize cytosolic Hsp70, could associate with CaM in the presence of 1 mM calcium. The synthetic peptide inhibited CaM-dependent NADK activity and PDE activity. This indicates that the 21-amino acid sequence at positions 261-281 is the CaM-binding site. The binding of CaM to Hsp70 inhibited the ATPase activity of Hsp70. The possible regulator function of Hsp70 in cell signaling events in response to heat stress is discussed.     

日本对虾(Marsupenaeys japonicus)HsP70基因cDNA的克隆与序列分析

根据中国明对虾(Fenneropenaeuschinensis)Hsp70的cDNA序列及日本对虾(Marsupenaeusjaponicus)Hsp70cDNA序列片段设计引物,用RT-PCR方法,从经热休克处理的日本对虾鳃总RNA中克隆到长1992bp的Hsp70cDNA序列,包括长1959bp的完整编码序列(CDS)。根据编码序列推导出相应的652个氨基酸,与其他真核生物Hsp70家族成员进行同源性比较,发现与凡纳滨对虾(Litopenaeusvannamei)、斑节对虾(PinaeusmonodonFabricius)、中国明对虾(Penaeuschinesis)的相似度分别为99.5%、99.4%、99.4%,与日本沼虾(Macrobrachiumnipponense)和罗氏沼虾(M.rosenbergii)的相似度分别为93.7%和92.9%,与虹鳟(Oncorhynchusmykiss)、原鸡(Callusgallus)、家鼠(Musmusculus)和人(Homosapiens)的相似度为89.2%、85.4%、88.3%和88.3%,表现出较高的保守性。分析表明所得到的日本对虾Hsp70是一种Dnak亚家族类型的细胞质Hsp70,拥有完整的N端ATP酶结构域(约45kDa)、底物肽结合结构域(18kDa)及C端结构域(10kDa)。以上结果说明我们所得到的序列是一条包含完整CDS的Hsp70基因序列。     

Intrinsic inhibition of the Hsp90 ATPase activity

The molecular chaperone Hsp90 is required for the folding and activation of a large number of substrate proteins. These are involved in essential cellular processes ranging from signal transduction to viral replication. For the activation of its substrates, Hsp90 binds and hydrolyzes ATP, which is the key driving force for conformational conversions within the dimeric chaperone. Dimerization of Hsp90 is mediated by a C-terminal dimerization site. In addition, there is a transient ATP-induced dimerization of the two N-terminal ATP-binding domains. The resulting ring-like structure is thought to be the ATPase-active conformation. Hsp90 is a slow ATPase with a turnover number of 1 ATP/min for the yeast protein. A key question for understanding the molecular mechanism of Hsp90 is how ATP hydrolysis is regulated and linked to conformational changes. In this study, we analyzed the activation process structurally and biochemically with a view to identify the conformational limitations of the ATPase reaction cycle. We showed that the first 24 amino acids stabilize the N-terminal domain in a rigid state. Their removal confers flexibility specifically to the region between amino acids 98 and 120. Most surprisingly, the deletion of this structure results in the complete loss of ATPase activity and in increased N-terminal dimerization. Complementation assays using heterodimeric Hsp90 show that this rigid lid acts as an intrinsic kinetic inhibitor of the Hsp90 ATPase cycle preventing N-terminal dimerization in the ground state. On the other hand, this structure acts, in concert with the 24 N-terminal amino acids of the other N-terminal domain, to form an activated ATPase and thus regulates the turnover number of Hsp90.     

Isolation and characterization of isoforms of HspBP1, inhibitors of Hsp70

The protein sequences derived from cDNA sequences for Hsp70 binding proteins from human (HspBP2) and rat tissues (HspBPR) are presented in this paper. The derived amino acid sequences of these proteins differ from human HspBP1 in the number of consecutive glycines near the amino-terminus. These differences, however, do not alter the inhibitory activity.     

Role of Hsp70 in cancer growth and survival

Hsp70 is a highly conserved protein that refolds misfolded proteins and has numerous housekeeping functions which regulate apoptosis and other cell activities. Hsp70 consists of a nucleotide binding domain which binds ATP and a substrate binding domain that binds misfolded proteins. The substrate binding domain contains a peptide binding pocket which is covered by a helical lid. In humans, there are three major cytosolic Hsp70 isotypes, Hsp70-8, Hsp70-1 and Hsp70-2. Leukemic and numerous other cancer cells have a greater amount of Hsp70-1 and -2, which help the cancer cells inhibit apoptosis in response to stress. This review summarizes the structure and role of Hsp70 proteins in cancer survival.     

ATPase domain of Hsp70 exhibits intrinsic ATP-ADP exchange activity

The chaperone activity of Hsp70 in protein folding and its conformational switching are regulated through the hydrolysis of ATP and the ATP-ADP exchange cycle. It was reported that, in the presence of physiological concentrations of ATP (approximately 5 mM) and ADP (approximately 0.5 mM), Hsp70 catalyzes ATP-ADP exchange through transfer of gamma-phosphate between ATP and ADP, via an autophosphorylated intermediate, whereas it only catalyzes the hydrolysis of ATP in the absence of ADP. To clarify the functional domain of the ATP-ADP exchange activity of Hsp70, we isolated the 44-kD ATPase domain of Hsp70 after limited proteolysis with alpha-chymotrypsin (EC 3.4.21.1). The possibility of ATP-ADP exchange activity of a contaminating nucleoside diphosphate kinase (EC 2.7.4.6) was monitored throughout the experiments. The purified 44-kD ATPase domain exhibited intrinsic ATP-ADP exchange by catalyzing the transfer of gamma-phosphate between ATP and ADP with acid-stable autophosphorylation at Thr204.     

Allosteric regulation of Hsp70 chaperones involves a conserved interdomain linker

The 70-kDa heat shock proteins (Hsp70) are essential members of the cellular chaperone machinery that assists protein-folding processes. To perform their functions Hsp70 chaperones toggle between two nucleotide-controlled conformational states. ATP binding to the ATPase domain triggers the transition to the low affinity state of the substrate-binding domain, while substrate binding to the substrate-binding domain in synergism with the action of a J-domain-containing cochaperone stimulates ATP hydrolysis and thereby transition to the high affinity state. Thus, ATPase and substrate-binding domains mutually affect each other through an allosteric control mechanism, the basis of which is largely unknown. In this study we identified two positively charged, surface-exposed residues in the ATPase domain and a negatively charged residue in the linker connecting both domains that are important for interdomain communication. Furthermore, we demonstrate that the linker alone is sufficient to stimulate the ATPase activity, an ability that is lost upon amino acid replacement. The linker therefore is most likely the lever that is wielded by the substrate-binding domain and the cochaperone onto the ATPase domain to induce a conformation favorable for ATP hydrolysis. Based on our results we propose a mechanism of interdomain communication.     

The Hsp70 peptide-binding domain determines the interaction of the ATPase domain with Tim44 in mitochondria

Ssc1, a molecular chaperone of the Hsp70 family, drives preprotein import into the mitochondrial matrix by a specific interaction with the translocase component Tim44. Two other mitochondrial Hsp70s, Ssc3 (Ecm10) and Ssq1, show high sequence homology to Ssc1 but fail to replace Ssc1 in vivo, possibly due to their inability to interact with Tim44. We analyzed the structural basis of the Tim44 interaction by the construction of chimeric Hsp70 proteins. The ATPase domains of all three mitochondrial Hsp70s were shown to bind to Tim44, supporting the active motor model for the Hsp70 mechanism during preprotein translocation. The peptide-binding domain of Ssc1 sustained binding of Tim44, while the peptide-binding domains of Ssc3 and Ssq1 exerted a negative effect on the interaction of the ATPase domains with Tim44. A mutation in the peptide-binding domain of Ssc1 resulted in a similar negative effect not only on the ATPase domain of Ssc1, but also of Ssq1 and Ssc3. Hence, the determination of a crucial Hsp70 function via the peptide-binding domain suggests a new regulatory principle for Hsp70 domain cooperation.     

N-Ethylmaleimide-modified Hsp70 inhibits protein folding.

Hsp70 molecular chaperones facilitate protein folding and translocation by binding to hydrophobic regions of nascent or unfolded proteins, thereby preventing their aggregation. N-Ethylmaleimide (NEM) inhibits the ATPase and protein translocation-stimulating activities of the yeast Hsp70 Ssa1p by modifying its three cysteine residues, which are located in its ATPase domain. NEM alters the conformation of Ssa1p and disrupts the coupling between its nucleotide- and polypeptide-binding domains. Ssa1p and the yeast DnaJ homolog Ydj1p constitute a protein folding machinery of the yeast cytosol. Using firefly luciferase as a model protein to study chaperone-dependent protein refolding, we have found that NEM also inhibits the protein folding activity of Ssa1p. Interestingly, the NEM-modified protein (NEM-Ssa1p) is a potent inhibitor of protein folding. NEM-Ssa1p can prevent the aggregation of luciferase and stimulate the ATPase activity of Ssa1p suggesting that it acts as an inhibitor by binding to nonnative forms of luciferase and by competing with them for the polypeptide binding site of Ssa1p. NEM-Ssa1p inhibits Ssa1p/Ydj1p-dependent protein refolding at different stages indicating that the chaperones bind and release nonnative forms of luciferase multiple times before folding is completed.     

Functional divergence between co-chaperones of Hsc70

The ATPase cycle of the chaperone Hsc70 is regulated by co-chaperones; Hsp40/DnaJ-related proteins stimulate ATP hydrolysis by Hsc70 and can bind unfolded polypeptides themselves. Conversely, various nucleotide exchange factors (NEFs) stimulate ADP-ATP exchange by Hsc70. We analyzed the purified Hsp40-related co-chaperones DJA1 (Hdj2) and DJA2 (Hdj3) and found that they had a distinct pattern of binding to a range of polypeptides. DJA2 alone could stimulate Hsc70-mediated refolding of luciferase in the absence of NEF, whereas DJA1 was much less active. The addition of the Bag1 NEF increased refolding by Hsc70 and DJA2, as did the newly characterized NEF Hsp110, but each NEF had a different optimal concentration ratio to Hsc70. Notably, the NEF HspBP1 could not increase refolding by Hsc70 and DJA2 at any concentration, and none of the NEFs improved the refolding activity with DJA1. Instead, DJA1 was inhibitory of refolding with DJA2 and Hsc70. All combinations of DJA1 or DJA2 with the three NEFs stimulated the Hsc70 ATPase rate, although Hsp110 became less effective with increasing concentrations. A chimeric DJA2 having its Hsc70-stimulatory J domain replaced with that of DJA1 was functional for polypeptide binding and ATPase stimulation of Hsc70. However, it could not support efficient Hsc70-mediated refolding and also inhibited refolding with DJA2 and Hsc70. These results suggest a more complex model of Hsc70 mechanism than has been previously thought, with notable functional divergence between Hsc70 co-chaperones.     

Modulation of chaperone activities of Hsp70 and Hsp70-2 by a mammalian DnaJ/Hsp40 homolog, DjA4

Type I DnaJs comprise one type of Hsp70 cochaperones. Previously, we showed that two type I DnaJ cochaperones, DjA1 (HSDJ/Hdj-2/Rdj-1/dj2) and DjA2 (cpr3/DNAJ3/Rdj-2/dj3), are important for mitochondrial protein import and luciferase refolding. Another type I DnaJ homolog, DjA4 (mmDjA4/dj4), is highly expressed in heart and testis, and the coexpression of Hsp70 and DjA4 protects against heat stress-induced cell death. Here, we have studied the chaperone functions of DjA4 by assaying the refolding of chemically or thermally denatured luciferase, suppression of luciferase aggregation, and the ATPase of Hsp70s, and compared these activities with those of DjA2. DjA4 stimulates the hydrolysis of ATP by Hsp70. DjA2, but not DjA4, together with Hsp70 caused denatured luciferase to refold efficiently. Together with Hsp70, both DjA2 and DjA4 are efficient in suppressing luciferase aggregation. bag-1 further stimulates ATP hydrolysis and protein refolding by Hsp70 plus DjA2 but not by Hsp70 plus DjA4. Hsp70-2, a testis-specific Hsp70 family member, behaves very similarly to Hsp70 in all these assays. Thus, Hsp70 and Hsp70-2 have similar activities in vitro, and DjA2 and DjA4 can function as partner cochaperones of Hsp70 and Hsp70-2. However, DjA4 is not functionally equivalent in modulating Hsp70s.     

The dual-specificity phosphatase hYVH1 interacts with Hsp70 and prevents heat-shock-induced cell death

hYVH1 [human orthologue of YVH1 (yeast VH1-related phosphatase)] is an atypical dual-specificity phosphatase that is widely conserved throughout evolution. Deletion studies in yeast have suggested a role for this phosphatase in regulating cell growth. However, the role of the human orthologue is unknown. The present study used MS to identify Hsp70 (heat-shock protein 70) as a novel hYVH1-binding partner. The interaction was confirmed using endogenous co-immunoprecipitation experiments and direct binding of purified proteins. Endogenous Hsp70 and hYVH1 proteins were also found to co-localize specifically to the perinuclear region in response to heat stress. Domain deletion studies revealed that the ATPase effector domain of Hsp70 and the zinc-binding domain of hYVH1 are required for the interaction, indicating that this association is not simply a chaperone-substrate complex. Thermal phosphatase assays revealed hYVH1 activity to be unaffected by heat and only marginally affected by non-reducing conditions, in contrast with the archetypical dual-specificity phosphatase VHR (VH1-related protein). In addition, Hsp70 is capable of increasing the phosphatase activity of hYVH1 towards an exogenous substrate under non-reducing conditions. Furthermore, the expression of hYVH1 repressed cell death induced by heat shock, H2O2 and Fas receptor activation but not cisplatin. Co-expression of hYVH1 with Hsp70 further enhanced cell survival. Meanwhile, expression of a catalytically inactive hYVH1 or a hYVH1 variant that is unable to interact with Hsp70 failed to protect cells from the various stress conditions. The results suggest that hYVH1 is a novel cell survival phosphatase that co-operates with Hsp70 to positively affect cell viability in response to cellular insults.     

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.