Sti1 Is a Non-competitive Inhibitor of the Hsp90 ATPase BINDING PREVENTS THE N-TERMINAL DIMERIZATION REACTION DURING THE ATPASE CYCLE*

The molecular chaperone Hsp90 is known to be involved in the activation of key regulatory proteins such as kinases, steroid hormone receptors, and transcription factors in an ATP-dependent manner. During the chaperone cycle, Hsp90 has been found associated with the partner protein Hop/Sti1, which seems to be required for the progression of the cycle. However, little is known about its specific function. Here we have inves-tigated the interaction of Sti1 from Saccharomyces cerevisiae with Hsp90 and its influence on the ATPase activity. We show that the inhibitory mechanism of Sti1 on the ATPase activity of Hsp90 is non-competitive. Sti1 binds to the N- and C-terminal part of Hsp90 and prevents the N-terminal dimerization reaction that is required for efficient ATP hydrolysis. The first 24 amino acids of Hsp90, a region shown previously to be important for the association of the N-terminal domains and stimulation of ATP hydrolysis, seems to be important for this interaction. a series of experiments in which the concentration of (P (cid:1) )MABA-ATP was varied, whereas the concentration of protein was left unchanged. The individual time traces were analyzed with single exponential equations. Replots of these series of experiments with the observed rate constant ( k obs ) as a function of ligand concentration followed straight lines, which is consistent with a simple, one-step binding mechanism. The rate constant for dissociation ( k off ) could be derived from the intercept and checked for consistency with the directly measured rate constant, whereas k on is represented by the slope. A replot of the observed amplitudes of the individual time traces versus concentration directly gives the dissociation constant ( K d ), which can be compared with the one derived from the kinetic constants ( k off / k on ). ATPase Activity— ATPase activities were measured using a regener-ating ATPase assay as described by Ali et al. (21). The assays were performed in 120- (cid:3) l cuvettes, and the reduction of NADH concentration was detected by the decrease of adsorbance at 340 nm, using an Amer- sham Biosciences 40/60 spectrophotometer. The temperature was set to 37 °C unless indicated otherwise. Assays were performed in 40 m M HEPES, pH 7.5, 5 m M MgCl 2 , and 2 m M ATP. KCl was added at the concentrations indicated using a 1 M KCl stock solution. Typical protein concentrations were 2.5 (cid:3) M for Hsp90 and up to 20 (cid:3) M for Sti1. To determine contaminating ATPase activities that could co-purify with Hsp90 or Sti1, radicicol, a specific inhibitor of the Hsp90 ATPase, was used at severalfold excess. The remaining ATPase activity in the pres- ence of radicicol was interpreted to be background and was subtracted from the total activity. Isothermal Titration Calorimetry— Isothermal titration calorimetry was performed using a MicroCal VP-ITC instrument (Microcal Inc., Northampton, MA). For the binding of AMP-PNP to Hsp90 or Hsp90 (cid:1) Sti1 complexes, 4 m M

The molecular chaperone Hsp90 is known to be involved in the activation of key regulatory proteins such as kinases, steroid hormone receptors, and transcription factors in an ATP-dependent manner. During the chaperone cycle, Hsp90 has been found associated with the partner protein Hop/Sti1, which seems to be required for the progression of the cycle. However, little is known about its specific function. Here we have investigated the interaction of Sti1 from Saccharomyces cerevisiae with Hsp90 and its influence on the ATPase activity. We show that the inhibitory mechanism of Sti1 on the ATPase activity of Hsp90 is non-competitive. Sti1 binds to the N-and C-terminal part of Hsp90 and prevents the N-terminal dimerization reaction that is required for efficient ATP hydrolysis. The first 24 amino acids of Hsp90, a region shown previously to be important for the association of the N-terminal domains and stimulation of ATP hydrolysis, seems to be important for this interaction.
The molecular chaperone Hsp90 1 is known to be involved in the activation process of signal transduction molecules, such as kinases and transcription factors, among others (1)(2)(3)(4). The essential in vivo function of Hsp90 in Saccharomyces cerevisiae involves ATP binding and hydrolysis of the nucleotide ATP (4,5). ATP hydrolysis by Hsp90 is thought to involve conformational changes that lead to the transient association of the N-terminal domains of the dimeric chaperone Hsp90 (6,7). These and additional structural rearrangements lead to the trapping of the ATP molecules inside the protein (8). In particular, the first 24 amino acids of Hsp90 are required to perform this "activation by dimerization" mechanism as they are thought to be swapped between the two N-terminal domains (9).
Studies performed in the context of the Hsp90-dependent steroid hormone receptors in higher eucaryotes have identified partner proteins, which are involved in the Hsp90-mediated activation process (10,11). Transient association of these proteins with the Hsp90 chaperone machinery leads to a chaperone cycle that involves complexes of defined composition. These complexes are called "early complex," "intermediate complex," and "mature complex" respectively (12). The substrate proteins have to pass through these complexes to become activated. The early complex consists of the molecular chaperones Hsp70, Hsp40, and the co-chaperone Hop/Sti1. After association with Hsp90, the intermediate complex is formed. Here Hsp90 is primarily associated with Hop/Sti1, which serves as an adapter protein between Hsp70 and Hsp90 (10). In the mature complex, the proteins of the intermediate complex are exchanged for the proteins p23 and one of the large prolyl isomerases (FKBP51, FKBP52, or Cyp40) (13,14). The details of this process, which ultimately leads to the activation of the substrate protein, are not known, but ATP hydrolysis by Hsp90 and Hsp70 is thought to play a crucial part in this process (15). The Hsp90 cycle seems to be evolutionary conserved since most of the Hsp90 partner proteins involved in this cycle are known to exist in yeast as well. In the yeast system, it had been shown that Hop/Sti1 acts as a potent inhibitor of the Hsp90 ATPase, and it has been speculated that this inhibition is achieved by blocking the ATP binding site (16). The prolylisomerase Cpr6, a homologue of Cyp40, was able to reverse this inhibition, suggesting that the two proteins compete for the same binding site on Hsp90 (16). These sites, which involve TPR (tetratrico peptide repeat) domains, were shown to be at the C-terminal end of Hsp90 (17,18). For the human protein Hop, the crystal structure of the TPR domain in complex with a peptide and additional biochemical data suggest that the interaction between Hop and Hsp90 involves only the last 10 amino acids of Hsp90 (19,20). Since the organization of the Hsp90 cycle critically depends on Sti1 as a central component, we decided to analyze the interaction between Sti1 and Hsp90 in detail. We show that the inhibition of Hsp90 by Sti1 is not achieved by blocking the access of nucleotide to its binding site but rather by restricting conformational changes of Hsp90 that are required for the hydrolysis reaction. Importantly, Sti1 binding involves multiple binding sites on Hsp90, including the first 24 amino acids.

MATERIALS AND METHODS
Materials-Radicicol was from Sigma. Geldanamycin was a kind gift of the Experimental Drug Division, NCI, National Institutes of Health, Bethesda, MD. All other chemicals were from Merck. Peptides were obtained from Dr. Susanne Modrow, University Regensburg, Regensburg, Germany.
Expression Constructs-Deletion mutants of yeast Hsp90 were generated using the plasmid pET28-HSP82, containing the full-length HSP82 gene of S. cerevisiae with an N-terminal His tag as a template (7). All PCR fragments were cloned into the pET28b vector, resulting in the constructs pET28b-⌬8-HSP82, pET28b-⌬16-HSP82, and pET28b-⌬24-HSP82. The ⌬MEEVD-mutant of Hsp90 has been generated using * This work was supported by grants from the Deutsche Forschungsgemeinschaft (to J. B. and J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Protein Expression and Purification-His-Hsp90 and its deletion mutants were expressed in the strain BL21 (DE3) codϩ (Stratagene, La Jolla, CA) at 37°C in LB Kan and induced with 1 mM isopropyl-thiogalactoside. Cells were lysed using a cell disruption system (Constant Systems, Warwick, UK). Protein purification was done according to the protocol described in Richter et al. (7). Proteins were stored in 40 mM HEPES, pH 7.5, 20 mM KCl at concentrations of 1.5 mg/ml to 9 mg/ml at Ϫ80°C. Mass spectrometry was used to verify the integrity and purity of the proteins. Purification of Sti1 and Cpr6 was achieved using essentially the same purification steps. Sti1 was stored at a protein concentration of 10 mg/ml, and Cpr6 was stored at a concentration of 11.5 mg/ml.
Stopped-flow Analysis-Stopped-flow measurements were performed with a HiTech SF-61 DX2 instrument in 40 mM HEPES, pH 7.5, 20 mM KCl, 5 mM MgCl 2 using an ATP-nucleotide that is specifically modified at the ␥-phosphate to carry the MABA-label. The excitation slit was set to 0.5 nm, the excitation wavelength was set to 296 nm to avoid nucleotide inner filter effects for tryptophan/MABA energy transfer (FRET), and emission was detected through a cut-off-filter of 418 nm. The temperature was set to 25°C unless indicated otherwise; concentrations indicated refer to the concentrations in the mixing chamber.
Dissociation rate constants were measured directly by displacement of a preformed Hsp90⅐(P␥)MABA⅐ATP complex with excess unlabelled ligand. The observed kinetics followed single exponential equations. Association rate constants were derived from a series of experiments in which the concentration of (P␥)MABA-ATP was varied, whereas the concentration of protein was left unchanged. The individual time traces were analyzed with single exponential equations. Replots of these series of experiments with the observed rate constant (k obs ) as a function of ligand concentration followed straight lines, which is consistent with a simple, one-step binding mechanism. The rate constant for dissociation (k off ) could be derived from the intercept and checked for consistency with the directly measured rate constant, whereas k on is represented by the slope. A replot of the observed amplitudes of the individual time traces versus concentration directly gives the dissociation constant (K d ), which can be compared with the one derived from the kinetic constants (k off /k on ).
ATPase Activity-ATPase activities were measured using a regenerating ATPase assay as described by Ali et al. (21). The assays were performed in 120-l cuvettes, and the reduction of NADH concentration was detected by the decrease of adsorbance at 340 nm, using an Amersham Biosciences 40/60 spectrophotometer. The temperature was set to 37°C unless indicated otherwise. Assays were performed in 40 mM HEPES, pH 7.5, 5 mM MgCl 2 , and 2 mM ATP. KCl was added at the concentrations indicated using a 1 M KCl stock solution. Typical protein concentrations were 2.5 M for Hsp90 and up to 20 M for Sti1. To determine contaminating ATPase activities that could co-purify with Hsp90 or Sti1, radicicol, a specific inhibitor of the Hsp90 ATPase, was used at severalfold excess. The remaining ATPase activity in the presence of radicicol was interpreted to be background and was subtracted from the total activity.
Isothermal Titration Calorimetry-Isothermal titration calorimetry was performed using a MicroCal VP-ITC instrument (Microcal Inc., Northampton, MA). For the binding of AMP-PNP to Hsp90 or Hsp90⅐Sti1 complexes, 4 mM AMP-PNP was used in the injection syringe. Protein concentrations were 15 M Hsp90 to determine the binding constant of AMP-PNP to Hsp90. To analyze AMP-PNP binding to the complex of Hsp90 and Sti1, 32 M Sti1 had been added to 15 M Hsp90 prior to the isothermal titration calorimetry experiment. The buffer used in the syringe and the cell was 40 mM HEPES, pH 7.5, 150 mM KCl, 5 mM MgCl 2 at 25°C. As the binding constant for these conditions has been measured to be below 1 M by SPR (22), this amount of Sti1 has been found to be sufficient to guarantee complete saturation of Hsp90 with Sti1. 40 injections of ligand solution were done to fully saturate the protein in the cell. Data analysis was performed with the Origin software package (Microcal Inc.).
Surface Plasmon Resonance Spectroscopy-SPR analysis was carried out with a BiaCore X instrument (BiaCore, Uppsala, Sweden). Hsp90 was coupled to the surface of a CM5 sensor chip using amine coupling. The coupling has been performed according to the manufacturer's instructions. About 1200 resonance units of Hsp90 were coupled to flow cell 2 of the chip, whereas flow cell 1 was activated and blocked to obtain similar surfaces. First direct binding was used to obtain information about the response of the chip to Sti1 and Cpr6 at different concentra-tions. As direct measurements are sensitive to matrix and chip artifacts, we employed an indirect approach. Using the nearly linear part of the binding curve, we applied identical concentrations of partner protein with varying concentrations of Hsp90 and fragments thereof. The observed competition between soluble Hsp90 and the chip surface led to a complete reduction of the binding signal at high concentrations of soluble Hsp90, showing the specificity of the observed SPR response. The data were analyzed according to Mayr et al. (22) to obtain binding constants for Hsp90/Hsp90 fragments and Sti1 or Cpr6. For the binding of ⌬8-Hsp90 and Hsp90 to Sti1 that has been performed at different buffer conditions to analyze the influence of ATP on the binding affinity, the running buffer and the buffer for the injection were varied according to the specific needs.

RESULTS
The Interaction between Hsp90 and Sti1 Is Salt-dependent-Sti1 has been known to inhibit the ATPase activity of Hsp90 (16), but the mechanistic aspects of this function have not been elucidated. To analyze the interaction between the two proteins, we determined the efficiency of the inhibition of Hsp90 by Sti1 at different KCl concentrations (Fig. 1). Inhibition of Hsp90 by Sti1 was found to be almost complete if a 6-fold excess of Sti1 is used at a KCl concentration of 80 mM. An increase in salt concentration resulted in a marked decrease in the inhibitory effect of Sti1 at temperatures of 37°C, but the effect of KCl was much less pronounced at lower temperature (data not shown). As higher concentrations of Sti1 still resulted in further inhibition of Hsp90, this decrease is not due to a reduced Sti1 Is a Non-competitive Inhibitor of Hsp90 -To investigate the mechanism of the Sti1-induced inhibition of the Hsp90 ATPase activity, we used steady-state kinetic approaches. Specifically, the analysis of the K m value of Hsp90 for ATP at different Sti1 concentrations should allow us to differentiate between the different modes of inhibition. As this methodology requires that the Hsp90 concentrations used are well below the dissociation constant of the Sti1⅐Hsp90 complex, we decided to use a KCl concentration of 200 mM for this approach. This implies a dissociation constant of about 10 M for the Hsp90⅐Sti1 complex. The Hsp90 concentration used was 4 M, and the Sti1 concentrations were 0, 8, and 16 M, respectively. The K m values obtained for the ATP hydrolysis reaction were not affected by the presence of Sti1 (Fig. 2). However, the maximum velocity of the ATPase reaction was reduced. This behavior is usually interpreted as non-competitive inhibition.
Nucleotide Binding to the Sti1⅐Hsp90 Complex Is Not Affected by Sti1-Non-competitive inhibition implies that nucleotide binding is not affected in the Hsp90⅐Sti1 complex when compared with Hsp90 alone. We therefore performed isothermal titration calorimetry experiments in which we measured the binding of AMP-PNP to Hsp90 or to a preformed Hsp90⅐Sti1 complex. The titration curves were found to be nearly identical, both resulting in binding constants of about 30 M (Fig. 3). These data clearly show that nucleotide binding to the Hsp90⅐Sti1 complex is still possible but leave the possibility that nucleotide binding might become rate-limiting in the case of the Sti1⅐Hsp90 complex. To test this, we determined the kinetics of the nucleotide interaction for Hsp90 and the Hsp90⅐Sti1 complex. To this end, MABA-ATP binding and displacement to Hsp90 were monitored in the absence or presence of Sti1. The displacement experiments clearly show that the interaction of MABA-ATP with Hsp90 and the Hsp90⅐Sti1 com-plex is similar (Fig. 4A). Evaluation of the rate constants revealed that the accessibility of the nucleotide binding site of Hsp90 is enhanced in the Sti1⅐Hsp90 complex. In addition, the dissociation kinetics suggest that release might be faster for the Sti1⅐Hsp90 complex when compared with Hsp90 alone, with dissociation rate constants increasing from 2 s Ϫ1 to 8 s Ϫ1 in the presence of Sti1. Compensating effects were obtained for the association rate constants (Fig. 4B). Therefore we suggest that the inhibition of the Hsp90 ATPase activity by Sti1 is not the result of blocking access of ATP to the binding site but rather the result of conformational changes that influence the ability of the enzyme to hydrolyze ATP, as usually observed in non-competitive inhibition.
Sti1 Binding to Hsp90 Involves C-terminal and N-terminal Binding Sites-The observation that the Sti1-mediated decrease in the turnover of the Hsp90 ATPase is the result of non-competitive inhibition raised questions about the mechanistic aspects of this interaction. Previously, it has been shown for human Hsp90 that Hop binds to the last 10 amino acids of this protein (20). Therefore we were interested to see whether these results could be extended to the yeast system. To address this question, we analyzed the interaction of Sti1 with Hsp90 by SPR spectroscopy. Hsp90 was immobilized on the surface of a CM5 sensor chip as described previously (22), and binding of Sti1 was detected based on the change in resonance units. Sti1 binding can be competed efficiently by the addition of Hsp90 to the injection solution. This allowed us to determine a dissociation constant of ϳ40 nM for the Hsp90⅐Sti1 complex, which is in agreement with previous data (22). As a control, we used the interaction between Hsp90 and Cpr6, which had been found to have a comparable affinity constant (22). Using this assay, we first analyzed an Hsp90 mutant lacking the last 5 amino acids, MEEVD. We could not detect binding of this mutant to Sti1 in the SPR assay, confirming the importance of this site for the interaction between TPR proteins and Hsp90 (Fig. 5A). To analyze whether this is the only site of interaction between the two proteins, we used peptides comprising either the last 7 or the last 21 amino acids of Hsp90 as competitors in the SPR analysis of the interaction between Hsp90 and Sti1. These peptides were found to compete with Sti1 binding to Hsp90, albeit at about 1000-fold higher concentrations when compared with Sti1, indicating that the affinity of these peptides to Sti1 is much lower than that of Sti1 to Hsp90. For Cpr6, we could measure a binding constant of 5 or 10 M, respectively, for the two peptides, also well above the binding constant of Cpr6 to Hsp90 (Fig. 5B). We further analyzed the interaction between these peptides and Sti1 using ATPase assays. No influence of the peptides on the ATPase activity of Hsp90 has been observed up to concentrations of 100 M (data not shown), implying that binding of Sti1 to Hsp90 is probably 1000-fold stronger than binding of the peptides to Sti1. These data suggest that although the C-terminal amino acids play an important role in the interaction between Hsp90 and the TPR proteins Sti1 and Cpr6, additional binding sites are required for high affinity binding between the two proteins.
To address this issue, we performed competition experiments in which we used N-terminal and C-terminal truncated fragments of Hsp90 and determined dissociation constants for their interaction with Sti1 and Cpr6. This approach should allow us to detect differences in the binding of the two TPR proteins to Hsp90 (Fig. 6). Surprisingly, in the case of Sti1, the affinity for Hsp90 is decreased by a factor of about 8, when the N-terminal 24 amino acids of Hsp90 were missing (Fig. 6A, Table I). This effect was only observed for Sti1 and not for Cpr6, indicating a major difference in the binding of the two proteins (Fig. 6B, Table I). Clearly, these results suggest that Sti1 interacts with the most C-terminal part of Hsp90 and, at the same time, with the N-terminal domain. The cooperative nature of these interactions may be the reason that no interaction of Sti1 to each of the individual binding sites could be obtained.
Sti1 Has a Reduced Ability to Inhibit the ATPase Activity of ⌬8-Hsp90 -Based on the SPR data (Table I), Sti1 has been found to bind with nearly the same affinity to ⌬8-Hsp90 as to wt-Hsp90. Compared with the other N-terminal deletion mutants, ⌬8-Hsp90 is the only protein that still has an ability to hydrolyze ATP. The specific ATPase activity of ⌬8-Hsp90 has been measured to be 1.7 min Ϫ1 (9), and we therefore attempted to analyze the interaction between ⌬8-Hsp90 and Sti1 using ATPase assays. Surprisingly, Sti1 was almost unable to inhibit the ATPase activity of this mutant under conditions used for the complete inhibition of Hsp90. An increase in the Sti1 concentration revealed that the effect seen is a result of weaker binding of Sti1 to ⌬8-Hsp90 (Fig. 7). This disagrees with the SPR assays made so far (Table I). These data point to a mechanistic defect in the interaction between Sti1 and ⌬8-Hsp90. This Hsp90-mutant has been described as forming N-terminal dimers with much higher affinity than wild type protein, but only in the presence of ATP (9). We therefore questioned the involvement of nucleotides in the observed interactions. To address this issue, we performed the SPR assay for Hsp90 and ⌬8-Hsp90 in the absence and presence of nucleotides. Although for Hsp90 no influence could be observed, whether 2 mM ATP or no nucleotide was included in the running and injecting buffer (Fig. 8A), for ⌬8-Hsp90, we observed a tighter binding to the nucleotide-free form (Fig. 8B). Thus, we conclude that the increased N-terminal dimerization in the presence of ATP is the result of the weaker binding of Sti1 to the ATP-bound form of ⌬8-Hsp90 as compared with the nucleotide-free form of ⌬8-Hsp90.

DISCUSSION
Partner protein binding to Hsp90 is one of the characteristics of the chaperone cycle required for the proper maturation of the Hsp90 substrate proteins. Many Hsp90 partner proteins have been identified using steroid hormone receptors as natural substrates (23). These studies resulted in the identification of the prolyl-isomerases FKBP51, FKBP52, and Cyp40, as well as in the identification of Hop/Sti1 and p23/Sba1 (cf. Ref. 24). We know that a sequence of different defined Hsp90-containing complexes is required for the activation process of the substrate (11), but the function of the partner proteins in the chaperone cycle is still largely unknown. In the case of Hop/Sti1, the partner protein mediates the interaction between Hsp70 and Hsp90 in early complexes, and the S. cerevisiae homologue FIG. 6. Binding of Sti1 and Cpr6 to Hsp90 deletion fragments. The SPR competition assay was also used to determine regions in Hsp90 that are responsible for high affinity binding of Sti1 and Cpr6. The results are plotted in Table I. A, binding of Hsp90 (q) and the fragments Hsp90 -262C (E) and ⌬24-Hsp90 () to Sti1. RU, resonance units. B, binding of Hsp90 (q) and the fragments Hsp90 -262C (E) and ⌬24-Hsp90 () to Cpr6.  7. Influence of Sti1 on ⌬8-Hsp90. ATPase assays were performed at identical conditions as in Fig. 1 to monitor the influence of Sti1 on the Hsp90 deletion mutant ⌬8-Hsp90 (q) as compared with Hsp90 (E). The data were analyzed using least square data analysis. inhibits the ATPase activity of Hsp90 (16). For the reconstruction and deconvolution of the chaperone cycle, it is of importance to understand these interactions in detail, including the mode of inhibition of the ATPase activity of Hsp90.
Our data show that the inhibition, which had been reported previously to result from the steric hindrance of ATP binding to the N-terminal domain of Hsp90, is in fact the result of a non-competitive inhibition. Nucleotide binding to Hsp90 occurs normally, even if Hsp90 is in a complex with Sti1. This observation prompted us to further investigate the binding properties of the two proteins. We identified a binding site for Sti1 in the last amino acids of yeast Hsp90, which agrees with data reported previously for hHsp90 and Hop (20). Here the Cterminal peptide was found to form a complex with the second TPR domain of Hop in which the peptide is completely surrounded by the ␣-helices of this domain. For the yeast protein, we were able to detect an additional Sti1 binding site in the N-terminal domain of Hsp90. Here, the deletion of the first 24 amino acids of Hsp90 resulted in an inhibition similar to that seen for the deletion of the whole N-terminal domain. This suggests that either the first amino acids directly serve as the N-terminal binding site or the deletion distorts neighboring regions, which form the additional docking site for Sti1. Our data do not allow us to discriminate between these two possibilities. However, we know that the overall structure of the N-terminal domain is not affected by the deletion (9).
Our findings concerning the binding of Sti1 to yeast Hsp90 may be valid also for high eucaryotes because here it was shown that deletion of parts of the N-terminal domain of Hsp90 results in a decrease of Hop binding (11). However, for the human Hsp90 system, no effect of Hop on the ATPase of hHsp90 has been observed (25). It could, however, well be that the inhibitory effect of Hop on Hsp90 is very sensitive to environmental conditions. Additionally, the ATPase activity of the human Hsp90 already is reduced by a factor of 20 when compared with the yeast Hsp90 (25). This may reflect evolutionary changes. Therefore there may be no need for Hop in the human system to act as an inhibitor of Hsp90.
The mechanism of regulation of Hsp90 by Sti1 seems to be the prevention of association of the N-terminal domains (Fig.  9). Using a deletion mutant that is ATPase-active and that had been shown to form N-terminal dimers upon addition of ATP even more pronounced than wt-Hsp90 (9), we observed a decreased inhibitory effect of Sti1. This decreased inhibitory effect is the result of a decreased affinity toward the ATPbound state as the binding to the nucleotide-free form occurs with nearly unchanged affinity. As it is known that the N-terminal domain is dimerized in the presence of ATP, it is reasonable to assume that Sti1 inhibits the N-terminal dimerization of Hsp90, thereby slowing the turnover of the ATPase to nearly undetectable levels. Thus, the binding of Sti1 to the N-terminal domain, maybe to the first 24 amino acids, seems to be the mechanism by which Sti1 inhibits the N-terminal dimerization.