(Received for publication, October 24, 1994; and in revised form, December 13, 1994)
From the
At normal temperatures, Hsp90 is one of the most abundant proteins in the cytosol of various eucaryotic cells. Upon heat shock, the level of Hsp90 is increased even more, suggesting that it is important for helping cells to survive under these conditions. However, studies so far have been almost exclusively concerned with the function of Hsp90 under non-stress conditions, and therefore only little is known about the role of Hsp90 during heat shock. As a model for heat shock in vitro, we have monitored the inactivation and subsequent aggregation of dimeric citrate synthase (CS) at elevated temperatures. Hsp90 effectively ``stabilized'' CS under conditions where the enzyme is normally inactivated and finally aggregates very rapidly. A kinetic dissection of the unfolding pathway of CS succeeded in revealing two intermediates which form and subsequently undergo irreversible aggregation reactions. Hsp90 apparently interacts transiently with these highly structured early unfolding intermediates. Binding and subsequent release of the intermediates favorably influences the kinetic partitioning between two competing processes, the further unfolding of CS and the productive refolding to the native state. As a consequence, CS is effectively stabilized in the presence of Hsp90. The significance of this interaction is especially evident in the suppression of aggregation, the major end result of thermal unfolding events in vivo and in vitro. These effects, which are ATP-independent, seem to be a general function of members of the Hsp90 family, since yeast and bovine Hsp90 as well as the Hsp90 homologue from Escherichia coli gave similar results. It seems likely that this function also reflects the role of Hsp90 under heat shock conditions in vivo. We therefore propose that members of the Hsp90 family convey thermotolerance by transiently binding to highly structured early unfolding intermediates, thereby preventing their irreversible aggregation and stabilizing the active species.
Members of the 90-kDa heat shock family are extremely abundant heat shock proteins under physiological conditions (Lindquist and Craig, 1988). Despite this fact, only specific interactions with a limited number of relatively non-abundant substrate proteins have been described, the best analyzed being the association of Hsp90 with unliganded steroid receptors and newly synthesized tyrosine kinases (for review, see Pratt, 1993; Jakob and Buchner, 1994). In addition, interactions with various other kinases and tubulin have also been detected (Sanchez et al., 1988; Miyata and Yahara, 1992; Xu and Lindquist, 1993). Recently, we presented evidence that Hsp90 may act as a general chaperone in protein folding at normal temperatures (Wiech et al., 1992). In vitro folding experiments showed that Hsp90 interacts with folding polypeptide chains. Hsp90 promoted functional refolding to the native state by suppressing unspecific side reactions. Further evidence for the chaperone activity of Hsp90 has been obtained for the folding of the muscle specific DNA-binding protein MyoD (Shaknovich et al., 1992; Shue and Kohtz, 1994). Given these in vitro results it is likely that Hsp90 is involved in folding events in vivo under non-stress conditions.
At elevated temperatures the concentration of Hsp90 increases severalfold, suggesting a protective or restoring role of Hsp90 under heat shock conditions (Lindquist, 1980; Nover, 1991; Parsell and Lindquist, 1994). In agreement with this notion, reactivation of thermally inactivated firefly luciferase is effectively stimulated by reticulocyte lysate and also by a purified system which contained Hsp90, Hsp70, and ATP (Schumacher et al., 1994).
In addition to its role in assisting refolding, we wondered
if Hsp90 may also influence the thermal unfolding process of
proteins in vitro. Mitochondrial citrate synthase (CS) ()was chosen as a model substrate because it is inactivated
and aggregates rapidly upon incubation at 43 °C. CS is a dimeric
protein composed of two identical 48 kDa subunits, which catalyzes the
reaction of oxaloacetic acid (OAA) and acetyl-CoA to form citric acid
and CoA. In the absence of the substrates, the midpoint of the thermal
unfolding transition of CS has been shown to be at 48.0 °C (Zhi et al., 1991). The change in structure is accompanied by a
loss in enzymatic activity. Binding of OAA and acetyl-CoA produces
large conformational changes in CS (Remington et al., 1982;
Wiegand et al., 1984; Karpusas et al., 1990) and
significant stabilization under thermal denaturing conditions (Wieland et al., 1964; Srere, 1966). A shift of T
from 48.0 to 66.5 °C in the presence of both substrates
has been observed (Zhi et al., 1991).
We have kinetically analyzed the thermal unfolding pathway of CS using ligand binding as a tool to identify unfolding intermediates. We demonstrated that Hsp90 binds transiently to unfolding intermediates of thermal unfolding CS. Upon release from Hsp90, the intermediates are able to refold rapidly to the native state. Hsp90 apparently stabilizes the native CS and slows dramatically the subsequent aggregation process.
The concentrations of yeast Hsp90 and E. coli Hsp90 were determined using the extinction coefficient of 0.72 and 1.25, respectively, for a 1 mg/ml solution in a 1-cm cuvette (Wetlaufer, 1962). The concentration of bovine Hsp90 was determined according to Bradford(1976) using BSA as a standard. For CS the published extinction coefficient of 1.78 for a 1 mg/ml solution in a 1-cm cuvette was used (West et al., 1990). CS was stored in 50 mM Tris, 2 mM EDTA, pH 8.0. Concentrations of CS and Hsp90 (Minami et al., 1994) are given for the dimeric form.
Figure 4:
Influence of Hsp90 on the reactivation
kinetics of citrate synthase at 43 °C. A, time course of
OAA-induced reactivation of CS in the presence of a 10-fold molar
excess of yeast Hsp90. The inactivation of CS (0.075 µM)
was performed at 43 °C in the presence of 0.75 µM yeast Hsp90. After 5 min of incubation, reactivation was initiated
by the addition of 1 mM OAA to the inactivation assay. At the
time points indicated, aliquots were taken and the enzymatic activity
was determined (). The reactivation reaction followed an apparent
first-order reaction (-) with a rate constant of k
= 6.1
10
s
Inset, after 5 min of
incubation of CS at 43 °C in the presence of Hsp90 (as indicated by
the arrow), either inactivation was continued (
) or
reactivation of CS was initiated by adding 1 mM OAA to the
inactivation reaction (
). The inactivation reaction followed a
first-order kinetic with k
= 1.7
10
s
. B, influence of
the Hsp90/CS ratio on the rate constants of the fast and slow refolding
reaction of CS. CS (0.075 µM) was incubated at 43 °C
in the presence of increasing amounts of yeast Hsp90. At the time point
where about 60% active citrate synthase was left, reactivation of CS
was initiated by adding OAA (final concentration, 1 mM). The
rate constants for the fast (
) and slow (
) refolding
reaction were obtained by analyzing the reactivation kinetics of CS
depending on the Hsp90-concentration used. Inset, influence of
the Hsp90/CS ratio on the relative amount of intermediates I
and I
represented by the amplitudes A
and
A
in percent. The amplitudes were calculated according to
the rate constants shown in the main part of the
figure.
Figure 2:
Inactivation and subsequent OAA-induced
reactivation of CS at 43 °C. A, influence of OAA on the
thermal inactivation of CS at 43 °C. The inactivation of CS (0.075
µM) was performed at 43 °C in the presence of 1.7
µM IgG. After 5 min of incubation at 43 °C (as
indicated by the arrow), either inactivation was continued
() or reactivation was initiated by adding 1 mM OAA to
the inactivation reaction (
). At the time points indicated,
aliquots were taken and the enzymatic activity was determined. The
inactivation reaction followed a first-order kinetics with k
= 7.4
10
s
. Inset, inactivation of CS (0.075
µM) in the absence (
) or presence (
) of 1
mM OAA at 43 °C. B, time course of OAA-induced
reactivation of CS. Correlation of the experimental data (
) with
single(- - -) and two exponential (-) fits. After 2 min of CS
incubation in the presence of 1.7 µM IgG at 43 °C,
OAA-induced reactivation was initiated and monitored as described under A. The reactivation reaction followed the two exponential fit
better with the two rate constants being k
= 50
10
s
, k
= 4.2
10
s
.
Figure 1:
Inactivation and subsequent aggregation
kinetics of citrate synthase (CS) at elevated temperatures. A, influence of Hsp90 on the thermal inactivation of CS. CS
(0.075 µM) was incubated at 43 °C in the presence of
0.075 µM Hsp90 (), 0.15 µM Hsp90
(
), 0.3 µM Hsp90 (
), 0.6 µM Hsp90
(
), 1.7 µM IgG (
), and in the absence of
additional protein (
). At the times indicated, aliquots were
withdrawn and the activity was determined as described. The solid
lines represent single exponential functions with apparent rate
constants given in the inset and in Table 1. Inset, influence of the Hsp90/CS ratio on the apparent rate
constant of the thermal inactivation of CS at 43 °C. The rate
constants were obtained from the inactivation kinetics shown in the
main part of the figure. B, influence of the temperature on
the thermal aggregation of CS. CS was diluted to a final concentration
of 0.075 µM at (
) 37 °C, (
) 40 °C,
or (
) 43 °C. No additional protein was present in the
incubation reaction. Aggregation was determined by light scattering. C, influence of Hsp90 from different sources on the thermal
aggregation process of CS at 43 °C. CS was diluted to a final
concentration of 0.075 µM in the presence of 0.15
µM yeast Hsp90 ± 1 mM MgATP (
),
0.15 µM bovine Hsp90 (
), 0.3 µME.
coli Hsp90 (
), 0.4 µM BSA (
), or 1 mM OAA (
). The kinetics of aggregation were determined by
light scattering. Inset, thermal unfolding of CS (0.075
µM) in the presence of 0.15 µM bovine Hsp90
or 0.4 µM BSA; the kinetics represent the change in
fluorescence at 336 nm.
The presence of stoichiometric amounts of yeast Hsp90 during the incubation of CS at 43 °C changed the inactivation kinetics significantly (Fig. 1A). An equimolar ratio of yeast Hsp90 to CS slowed down the inactivation process by 50%. In the presence of an 8-fold molar excess of Hsp90, the obtained apparent rate constant of inactivation decreased 5-fold (Table 1). Thus, this effect was strongly dependent on the Hsp90/CS ratio (Fig. 1A, inset). Similar results were obtained with bovine and E. coli Hsp90 (Table 1). In control experiments IgG and BSA were added to a 23- and 16-fold molar excess, respectively. Only minor changes in the inactivation kinetics were observed (Fig. 1A, Table 1), showing that the stabilizing effect of Hsp90 is not simply due to additional protein being present during the time course of inactivation.
The presence of Hsp90 also affected the aggregation behavior of CS at 43 °C (Fig. 1C). A 2-fold molar excess of either yeast or bovine Hsp90 or slightly higher amounts of E. coli Hsp90 were sufficient to suppress the thermal aggregation of CS. BSA, again used as a control for unspecific protein effects, showed no significant influence. In contrast to Hsp70 and GroEL whose actions are ATP dependent, addition of ATP shows no detectable effect on the function of eu- and procaryotic Hsp90s in slowing the aggregation process of CS (Fig. 1C and data not shown).
The influence of Hsp90 on the overall unfolding/aggregation reaction of CS could also be monitored by following changes in intrinsic fluorescence of CS (Fig. 1C, inset). In the absence of Hsp90 a strong decrease in the fluorescence of CS was observed during the first 2 min of incubation at 43 °C. This was followed by an increase in fluorescence, indicating CS aggregation. The addition of Hsp90 again resulted in significant differences in the kinetics of CS unfolding. The initial decrease in fluorescence was slower, and no subsequent increase in fluorescence could be detected. This indicated that insignificant aggregation of CS occurred in the presence of the heat shock protein during the time course of the experiment.
Figure 3:
Analysis of intermediates on the CS
thermal unfolding pathway. CS was incubated at 43 °C in the
presence of 1.7 µM IgG. At the time points indicated,
aliquots were withdrawn and the activity was measured (). The
apparent rate constant obtained was k
=
7.4
10
s
. At the same
time reactivation of inactive CS molecules was initiated by adding OAA
(final concentration, 1 mM). After further incubation for 60
min at 43 °C, the activity of the reactivated samples was
determined. The amounts of intermediates were obtained by subtracting
the percentage of native species at the time points, at which the
reactivation was initiated, from the total amount of active species
determined after the end of the reactivation process (
). Open
squares (
) represent the calculated amount of irreversibly
denatured species.
The
reactivation reaction of CS could be described by two first-order
reactions with a fast phase (k = 50
10
s
) and a slower
refolding reaction with a rate constant of k
= 4.2
10
s
(Fig. 2B). In addition, Fig. 2B shows the correlation of a single and a two-exponential fit with
the experimental data. A single exponential fit could not represent the
experimental data adequately. A possible explanation for this biphasic
reactivation kinetic is the formation of at least two unfolding
intermediates I
and I
. Intermediate
I
, which is in a rapid equilibrium with the native state
and I
which is in a slower equilibrium with I
(see ``Discussion''). Due to the presence of both
intermediates and the distinct rate constants of the individual
refolding reactions, we could apply parallel reactivation mechanisms to
distinguish between intermediate I
and I
. We
analyzed the amplitudes of the individual reactivation reactions
initiated by addition of OAA at different time points during the
thermal inactivation of CS. Within the first 2 min of inactivation,
reactivation kinetics were dominated by the renaturation of the fast
refolding species intermediate I
(Table 2). After
longer inactivation periods, the fraction of slow refolding CS
(intermediate I
) increased. This accumulation of I
within the first 2 min of thermal inactivation implied that the
formation of I
is slightly faster than the conversion from
I
to I
. That no significant reactivation could
be detected within the first 75 s might be due to adsorption of
intermediate I
to the surface of the reaction vessel or
denatured CS which does not aggregate or reactivate.
The data presented here provide evidence that Hsp90 ``chaperones'' protein unfolding under heat shock conditions in vitro. Irreversible aggregation of proteins is the major reaction occurring upon heat shock in the cell (Pelham, 1985; Bensaude et al., 1990; Gragerov et al., 1991). Cells respond to the accumulation of unfolding and aggregating proteins by increasing the synthesis of several Hsps. In most eucaryotic cells, three classes of heat shock proteins, Hsp90, Hsp70, and small Hsps, are predominantly synthesized (Lindquist, 1980; Nover, 1991). Co-localization of Hsps with aggregates formed upon heat shock in vivo suggests that these proteins are involved in the prevention of intracellular aggregation (Bensaude et al., 1990). While the role of Hsp70 as a chaperone in this context was established early (Pelham, 1985), the function of Hsp90 under heat shock has remained enigmatic. Here we demonstrate that the presence of Hsp90 during the incubation of CS at elevated temperatures has significant effects both on inactivation kinetics as well as on the subsequent aggregation process. These effects are comparable to the effects of GroEL (data not shown) and small heat shock proteins on CS inactivation (Jakob et al., 1993). While CS is almost completely inactivated after 10 min of incubation at 43 °C in the absence of Hsp90, almost 40% of active molecules are still present if CS is incubated the same period in the presence of an 8-fold molar excess of yeast Hsp90. Similar results could be obtained using Hsp90 from bovine pancreas and E. coli. Control experiments using similar or even higher concentrations of BSA had no influence on the inactivation kinetics.
Early manifestations of structural changes in CS are accompanied by the loss of enzymatic activity. These minor changes are followed by irreversible aggregation processes due to the increasing influence of intra- and intermolecular hydrophobic interactions at higher temperatures (Jaenicke, 1987). We examined if Hsp90 exerts an influence not just on the initial inactivation of CS but also on the subsequent aggregation process by performing light scattering measurements either in the absence or presence of this heat shock protein. We found stoichiometric amounts of Hsp90 were able to slow down the aggregation process of CS significantly. About the same amount of Hsp90 was required to suppress aggregation as was required for the ``stabilization'' during inactivation. Hsp90 from different sources exerted the same suppression of aggregation, whereas BSA did not change the aggregation kinetics. These effects seem therefore to reflect general properties of members of the Hsp90 family. Unlike GroEL and Hsp70, whose actions require ATP, the function of Hsp90 is ATP-independent.
To elucidate the mechanism of Hsp90 action, an
analysis of the thermal unfolding pathway of CS seemed necessary. The
stabilizing effect of the substrate OAA was used as a tool to elucidate
a kinetic description for the first steps in this thermal unfolding
pathway. In the absence of OAA at 43 °C, the inactivation of CS
proceeded with a half-time of 1.7 min. The inactivation, which
apparently followed first-order kinetics, was only slightly dependent
on the CS concentration used. The concentration dependence observed
might be due to a subsequent aggregation process, observable by light
scattering. After incubation of CS at 43 °C for 2 min, measurable
aggregates could be detected. However, when OAA was added, neither
inactivation nor aggregation of CS was observed within the time range
of the experiment (Fig. 2A, inset, and
1C). These results were in good agreement with experiments by
Srere and co-workers, who showed that the formation of the
CSOAA complex results in a significantly higher
thermostability of CS (Zhi et al., 1991). Next, we
investigated how addition of OAA after start of the thermal
inactivation affects CS. We could show that the substrate not only
stabilized the still native molecules but also led to the reactivation
of about 30% of already inactive molecules. Since inactivation of CS
might be due to early manifestations of structural changes at the
active site (Zhi et al., 1992), it is unlikely that direct
binding of OAA to the inactive intermediates triggers their functional
refolding. We therefore suggest an equilibrium between native CS and
early unfolding intermediates, where the interaction of active CS with
OAA will shift the equilibrium toward the native state. As a
consequence the refolding of already inactive molecules can occur (). The reactivation kinetics could be explained by two
consecutive first-order reactions with a fast and a slow refolding
reaction. Thus the existence of at least two unfolding intermediates is
proposed. Intermediate I
refolds rapidly to the native
state (k
= 50
10
s
) and intermediate I
, which
refolds with significantly slower rate constant of k
= 4.2
10
s
.
Our experimental data and the thermal transition measurements performed
by Srere and co-workers(1991) suggest that both intermediates undergo
only unimolecular rearrangements, while remaining in a dimeric
conformation. We determined the concentrations of the intermediates
I
and I
during the inactivation process by
analyzing the amplitudes of the individual refolding reactions,
initiated at different time points after start of the incubation at 43
°C. The accumulation of intermediate I
within the first
2 min of inactivation indicated that the unfolding to I
is
faster than the following unimolecular rearrangement to I
and is also faster than the refolding to the native state (k
> k
and k
> k
). The thermal
unfolding pathway of CS can be described by the following
model:
where N-OAA is the native enzyme complexed with oxaloacetic
acid, N is the native state, I and I
are the
dimeric intermediates, and A are aggregates (k
> k
, k
> k
).
Upon incubation at elevated temperatures in
the absence of OAA, the native enzyme unfolds rapidly to intermediate
I, which is in a fast equilibrium with the native state.
Intermediate I
is in slow equilibrium with I
which can undergo an irreversible unfolding and/or aggregation
reaction. This irreversibility shifts the overall equilibrium toward
the aggregates A (k
> k
).
In the presence of OAA, most of the native enzyme is complexed with the
substrate. This complex provides higher thermostability, and thus CS is
not inactivated at these temperatures. When OAA is added during the
inactivation process, native CS molecules are complexed. As a
consequence, intermediate I
refolds rapidly to the native
state, with k
apparently >k
. Since the concentration of free N becomes
very small, the refolding of I
to N is also essentially
irreversible. The refolding of intermediate I
is limited by
the subsequent unfolding/aggregation reaction with the rate constant k
.
We could now address the question of the
underlying mechanism by which Hsp90 slows the thermal inactivation and
subsequent aggregation process of CS. We concluded that transient
interactions of Hsp90 with the substrate protein play a major part in
regulating the inactivation kinetics of CS. If Hsp90 binds irreversibly
to folding intermediates, no positive kinetic effect on slowing the
thermal inactivation would be expected. Interestingly, this result was
obtained in studies on the effect of GroEL on thermally denaturing
-glucosidase (Höll-Neugebauer et al.,
1992). GroEL formed stable complexes with the thermal unfolding
intermediates, thereby keeping the molecules inactive. As a
consequence, no GroEL-dependent deceleration in the rate of thermal
inactivation could be observed. On the other hand, transient
interactions of GroEL and thermal unfolding rhodanese resulted in a
significant increase in the half-time of inactivation (Mendoza et
al., 1992), comparable to the results presented here with Hsp90
and CS. This ``thermoprotective'' effect is due to transient
interactions of GroEL with rhodanese conformers, which are native-like
or are able to renature in the test assay (Mendoza et al.,
1992). That Hsp90 interacts transiently with CS unfolding intermediates
was further confirmed by performing reactivation studies of thermally
unfolding CS in the presence of Hsp90. If OAA was added after the start
of the inactivation, part of the inactive molecules could refold to the
native state. If partially unfolded CS molecules were irreversibly
bound to Hsp90, no reactivation would be expected. Additional evidence
for the transient character of the Hsp90 interaction came from
refolding studies on chemically denatured proteins in the presence of
Hsp90 (Wiech et al., 1992) as well as studies on the
helix-loop-helix protein MyoD (Shaknovich et al., 1992). Both
reports describe a transient interaction of Hsp90 with substrate
proteins. Also in the case of progesterone receptor-Hsp90 interactions
steady state assembly and disassembly cycles have been demonstrated
(Smith, 1993). Taking these results together we propose that isolated
Hsp90 is mainly engaged in transient association-dissociation reactions
with non-native substrate proteins. Whenever stable complexes between
Hsp90 and substrate proteins (steroid receptors, kinases) could be
detected, partner proteins like Hsp70, Hsp56, p60, or p23 were also
part of the complex (Pratt, 1993; Stancato et al., 1993; for
review see Jakob and Buchner, 1994), and ATP was required for
dissociation (Schumacher et al., 1994). The complexes of
isolated Hsp90 with substrate proteins, however, are intrinsically
unstable, and do not require ATP to trigger dissociation.
The next
question we asked was how Hsp90 exerts its function in
``stabilizing'' CS. Therefore, we examined the OAA-induced
refolding kinetics of CS in the presence of Hsp90 at high temperature.
In the presence of a 10-fold molar excess of Hsp90, only one apparent
rate constant could be detected which was comparable to the rate
constant of the slow refolding reaction of CS in the absence of
additional protein. Reactivation experiments in the presence of lower
amounts of Hsp90 led to reactivation kinetics which followed two
first-order reactions. The fast rate constant was still smaller than
the rate constant of the fast refolding reaction of I in
the absence of Hsp90 while the slow refolding reaction approached the
refolding rate of intermediate I
. We suggest that Hsp90
interacts transiently with intermediate I
and
I
, since both refolding reactions were slower in the
presence of Hsp90. Furthermore, we conclude that after initiating the
refolding reaction by adding OAA in the presence of Hsp90, the rate of
refolding of intermediate I
is slowed by the association
and dissociation of Hsp90 with I
. Upon refolding of
intermediate I
to I
again, the kinetic
competition between binding to Hsp90 and refolding to the native state
determines the rate constants. Therefore, the rate constants observed
mirror the overall refolding reaction of intermediate I
and
I
influenced by the binding to and release from Hsp90.
Although the interaction of Hsp90 with I
and I
and the subsequent suppression of aggregation has been clearly
demonstrated, these effects alone cannot be the entire reason for the
observed deceleration of the inactivation kinetics. Kinetic simulations
(using the program KinSim) suggest that a further, still enzymatically
active unfolding intermediate may exist, which is stabilized by
interaction with Hsp90. The existence of this intermediate cannot be
demonstrated using the techniques employed in this study. However, such
``native-like'' structures which possess enzymatic activity
but differ significantly from the crystal structure are not without
precedence (Cioni et al., 1994). Our proposal that Hsp90
interacts with highly structured unfolding intermediates seems to be
reminiscent of its function in the ``activation'' of steroid
receptors and certain kinases (Pratt, 1993; Smith, 1993). Hsp90 seems
to bind to these molecules when they are native-like but not in a fully
active conformation since they either lack interactions with ligands
(in the case of the receptors) or with membranes (in the case of
kinases).
Despite the interaction of unfolding intermediates with
Hsp90, part of I will unfold irreversibly, mainly dependent
on the rate constant k
. This explains why in the presence
of Hsp90 a slow increase in light scattering could still be detected.
Based on these findings we propose the following model:
We draw the conclusion that Hsp90 binds transiently to very
early, dimeric unfolding intermediates, therefore allowing the
stabilizing of still active species and functional refolding of I and I
to the native state by keeping the amount of
aggregation-sensitive intermediates low.
Taking all these results together and taking into account what is known about the function of Hsp90 in vivo, we propose that structured folding intermediates are the preferential substrates for Hsp90. In many studies reported so far, Hsp90 seems to interact with structured, inactive proteins, missing only the final conversion to the native state (Shue and Kohtz, 1994; Smith, 1993). Recently, this could also be shown for Grp94, the Hsp90 homologue from the endoplasmic reticulum. On the folding pathway of immunoglobulins, Grp94 interacts with late folding intermediates, which are already oxidized, fully folded but not yet assembled (Melnick et al., 1994). The action of Hsp90 in binding to highly structured folding intermediates appears to be rather different from the action of two other chaperones Hsp70 and GroEL which bind to peptide sequences and more disordered folding intermediates, respectively (cf. Hendrick and Hartl, 1993).
In the case of Hsp90 it seems possible, that the folding-unfolding rate of polypeptides determines the interaction with Hsp90. This would give a rational explanation for the ability of Hsp90 to interact with unrelated substrates (Jakob and Buchner, 1994).