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INTRODUCTION |
The molecular chaperone
Hsp901 is known to be
involved in the activation process of signal transduction molecules,
such as kinases and transcription factors, among others (1-4). 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.
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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 mutagenized primers. The other fragments
mentioned in the text are as described in Richter et al.
(7). pRSET-Cpr6 and pRSET-Sti1 are as described by Prodromou et
al. (16).
Protein Expression and Purification--
His-Hsp90 and its
deletion mutants were expressed in the strain BL21 (DE3) cod+
(Stratagene, La Jolla, CA) at 37 °C in LBKan 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
80 °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
MgCl2 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 Hsp90·(P
)MABA·ATP 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 (kobs) as a function of ligand concentration
followed straight lines, which is consistent with a simple, one-step
binding mechanism. The rate constant for dissociation
(koff) could be derived from the intercept and
checked for consistency with the directly measured rate constant,
whereas kon is represented by the slope. A
replot of the observed amplitudes of the individual time traces
versus concentration directly gives the dissociation
constant (Kd), which can be compared with the one
derived from the kinetic constants (koff/kon).
ATPase Activity--
ATPase activities were measured using a
regenerating ATPase assay as described by Ali et al.
(21). The assays were performed in 120-µl 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
MgCl2, and 2 mM ATP. KCl was added at the concentrations indicated using a 1 M KCl stock solution.
Typical protein concentrations were 2.5 µM for Hsp90 and
up to 20 µM 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
Hsp90·Sti1 complexes, 4 mM AMP-PNP was used in the
injection syringe. Protein concentrations were 15 µM
Hsp90 to determine the binding constant of AMP-PNP to Hsp90. To analyze
AMP-PNP binding to the complex of Hsp90 and Sti1, 32 µM
Sti1 had been added to 15 µM 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 MgCl2 at 25 °C. As
the binding constant for these conditions has been measured to be below
1 µM 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 concentrations. 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.
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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 inhibitory effect, but more
likely is the result of the weaker binding of Sti1 to Hsp90. The
apparent binding constants of Sti1 and Hsp90 changed from below 1 µM at 80 mM KCl to 20 µM at 250 mM KCl.

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Fig. 1.
Influence of KCl on the inhibition of Hsp90
by Sti1. Different concentrations of Sti1 were added to Hsp90 at
various KCl concentrations, and the resulting ATPase activities were
measured at 37 °C. The final concentrations of KCl were 80 ( ), 150 ( ), and 200 mM ( ). The data points were
analyzed using least square data analysis to obtain apparent binding
constants for the Sti1·Hsp90 complexes.
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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 Km 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 Sti1·Hsp90 complex, we decided to use a KCl
concentration of 200 mM for this approach. This implies a
dissociation constant of about 10 µM for the Hsp90·Sti1
complex. The Hsp90 concentration used was 4 µM, and the
Sti1 concentrations were 0, 8, and 16 µM, respectively.
The Km 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.

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Fig. 2.
Influence of Sti1 on the
Km-value of ATP binding to Hsp90. ATPase
activities were measured at varying ATP concentrations for preformed
Hsp90·Sti1 complexes. Hsp90 concentrations were 4 µM,
whereas for Sti1, the concentrations were 0 ( ), 8 ( ), and 16 µM ( ). The activities were measured at 200 mM KCl in the ATPase buffer to prevent complete formation
of complexes. The resulting activities were analyzed using least square
data analysis.
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Nucleotide Binding to the Sti1·Hsp90 Complex Is Not Affected by
Sti1--
Non-competitive inhibition implies that nucleotide binding
is not affected in the Hsp90·Sti1 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 Hsp90·Sti1 complex. The titration curves were found to be
nearly identical, both resulting in binding constants of about 30 µM (Fig. 3). These data
clearly show that nucleotide binding to the Hsp90·Sti1 complex is
still possible but leave the possibility that nucleotide binding might
become rate-limiting in the case of the Sti1·Hsp90 complex. To test
this, we determined the kinetics of the nucleotide interaction for
Hsp90 and the Hsp90·Sti1 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 Hsp90·Sti1 complex is similar (Fig.
4A). Evaluation of the rate
constants revealed that the accessibility of the nucleotide binding
site of Hsp90 is enhanced in the Sti1·Hsp90 complex. In addition, the
dissociation kinetics suggest that release might be faster for the
Sti1·Hsp90 complex when compared with Hsp90 alone, with dissociation
rate constants increasing from 2 s
1 to 8 s
1
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.

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Fig. 3.
Calorimetric analysis of AMP-PNP binding to
Hsp90 and preformed Hsp90·Sti1 complexes. Isothermal titration
calorimetry experiments were performed at 25 °C in the standard
ATPase buffer. The Hsp90 concentration was 15 µM in both
experiments, whereas in the second experiment, 32 µM Sti1
was additionally present. Titrations were performed with 35 injections
of 8 µl each and a concentration of 4 mM AMP-PNP in the
injection syringe. The data analysis was performed with the Origin
package included in the instrument software.
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Fig. 4.
Kinetics of MABA-ATP binding to Hsp90 and
Hsp90·Sti1 complexes. MABA-ATP bound to Hsp90 was recorded using
FRET between the MABA-ATP and tryptophan residues near the
binding site of MABA-ATP. A, displacement of MABA-ATP from
Hsp90 and Hsp90·Sti1 complexes. 2.5 µM Hsp90 was used
either alone ( ) or in complex with 5 µM Sti1 ( ).
B, kon-koff
analysis of MABA-ATP binding to Hsp90 and Hsp90·Sti1 complexes. 2.5 µM Hsp90 was used either alone ( ) or in complex with 5 µM Sti1 ( ). Data points were obtained by varying the
MABA-ATP concentration upon binding to the proteins and plotting the
observed rate constants against the MABA-ATP concentration.
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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 ~40 nM for the Hsp90·Sti1 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 µM, 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 µM (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.

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Fig. 5.
Sti1 binding to the C
terminus of Hsp90. As shown in A, SPR was used to
compare the binding of Hsp90 and MEEVD-Hsp90 to Sti1. Sti1 was used
at concentrations of 100 nM. Attempts to reduce the
resonance signal of Sti1 by adding Hsp90 or MEEVD-Hsp90 resulted in
the plotted effects of these proteins on the binding of Sti1 to the
chip surface. Cpr6 gave similar results (data not shown).
RU, resonance units. As shown in B, similar
experiments were performed with peptides derived from the C-terminal
sequence of Hsp90. The observed resonance signals were analyzed using
least square data analysis and resulted in binding constants of 4.2 ( ) and 10 µM ( ) for the peptides with 21 and 7 amino acids and Cpr6, respectively, and about 150 ( ) and 300 µM ( ) for the peptides to Sti1.
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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.

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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 ( ) and the fragments Hsp90-262C ( ) and 24-Hsp90 ( )
to Sti1. RU, resonance units. B, binding of Hsp90
( ) and the fragments Hsp90-262C ( ) and 24-Hsp90 ( ) to
Cpr6.
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Table I
Binding constants for the different Hsp90 fragments to Cpr6 and Sti1
The binding data were determined by SPR analysis as described under
"Materials and Methods."
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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
1 (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.

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Fig. 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 ( ) as compared with Hsp90
( ). The data were analyzed using least square data analysis.
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Fig. 8.
Effect of ATP on the affinity of Sti1 for
Hsp90 and 8-Hsp90. SPR competition assays
were performed with ( ) and without ( ) of 2 mM ATP,
1.5 mM MgCl2 in the standard ATPase buffer at
25 °C. A, the effect of ATP on the binding of Hsp90 to
Sti1. RU, resonance units. B, the effect of ATP
on the binding of 8-Hsp90 to Sti1.
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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 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 C-terminal
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 ATP-bound
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.

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Fig. 9.
Model of the inhibitory effect of Sti1 on the
Hsp90 ATPase. The model combines data from this and
previous studies (9, 16) on the Hsp90·Sti1 interaction. Sti is
depicted in gray. The schematic highlights the interaction
of Sti1 with the C- and N-terminal parts of the Hsp90 dimer.
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