From the Department of Biochemistry and Molecular Biology, Mayo Graduate School, Rochester, Minnesota 55906
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ABSTRACT |
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The chaperone hsp90 is capable of binding and
hydrolyzing ATP. Using information on a related ATPase, DNA gyrase B,
we selected three conserved residues in hsp90's ATP-binding domain for
mutation. Two of these mutations eliminate nucleotide binding, while
the third retains nucleotide binding but is apparently deficient in ATP
hydrolysis. We first analyzed how these mutations affect hsp90's binding to the co-chaperones p23 and Hop, and to the hydrophobic resin,
phenyl-Sepharose. These experiments showed that ATP's effects, specifically, increased affinity for p23 and decreased affinity for Hop
and phenyl-Sepharose, are brought on by ATP binding alone. We also
tested the ability of hsp90 mutants to assist hsp70, hsp40, and Hop in
the refolding of denatured firefly luciferase. While hsp90 is capable
of participating in this process in a nucleotide-independent manner,
the ability to hydrolyze ATP markedly potentiates hsp90's effect.
Finally, we assembled progesterone receptor heterocomplexes with hsp70,
hsp40, Hop, p23, and wild type or mutant hsp90. While neither ATP
binding nor hydrolysis was necessary to bind hsp90 to the receptor,
mature complexes containing p23 and capable of hormone binding were
only obtained with wild type hsp90.
The 90-kDa heat shock protein
(hsp90)1 is an abundant and
highly conserved protein involved in a diverse array of cellular processes. Its fundamental importance is underscored by its presence in
all species studied, from Escherichia coli to humans, with a
remarkable 40% amino acid identity (1, 2). Additionally, the level of
hsp90 expressed in various human and murine tissues represents up to
2% of total protein (3), and deletion studies in yeast have shown that
hsp90 is essential for viability (4). The common thread in many of its
known activities is the chaperoning of substrate proteins to activate
their function. This has been most extensively studied with steroid
receptors, where hsp90 is required to fold the hormone-binding domain
of these receptors into a conformation with high affinity for steroid
(see Ref. 5 for a review). In this process, hsp90 acts in protein
heterocomplexes with a number of other proteins, including hsp70,
hsp40, and the co-chaperones Hop and p23 (6-8). Heterocomplex
formation as well as hsp90/co-chaperone interaction are known to be
nucleotide-regulated (9-11). Hsp90 also participates in a more general
protein folding process with hsp70, hsp40, and Hop which requires
nucleotides (9, 12), and it can hold and stabilize denatured proteins for subsequent refolding by hsp70/hsp40 (13) or GroEL/ES (14). On its
own, hsp90 can bind proteins (15-19), peptides (18, 19), and
hydrophobic resins (11, 20, 21), but the effect of nucleotides on these
activities is variable and may depend upon the substrate involved.
Recently, clear evidence in the form of biochemical (22-24) and
crystallographic (25) studies has been presented demonstrating nucleotide binding to hsp90. This binding occurs in the
NH2-terminal domain of hsp90 at the same site as
geldanamycin binding (22, 25, 26). Geldanamycin is a specific inhibitor
of hsp90, known to disrupt a number of hsp90-dependent
processes, including activation of the oncogenic tyrosine kinase
pp60vsrc (27), steroid receptor hormone binding
(28, 29) and translocation (30), regulation of the HSF1 heat shock
transcription factor (31, 32), Cdc37-mediated stabilization of the
cyclin-dependent kinase Cdk4 (33), refolding of denatured
firefly luciferase in reticulocyte lysate (12, 34), and
ribonucleoprotein complex formation between hepatitis B virus reverse
transcriptase and its RNA primer (35). The effect of geldanamycin on
such a wide range of experimental systems points to the importance of
nucleotides in hsp90 activity.
Two recent studies have shown that mutant hsp90 proteins deficient in
either nucleotide binding or hydrolysis are incapable of supporting
growth in yeast (36, 37). In this paper, we use similar mutants to
first analyze their effects on hsp90's interactions with nucleotide
and hydrophobic resins, and with the co-chaperones p23 and Hop. Then,
we show how alterations in these individual interactions affect the
assembly and function of higher-order protein heterocomplexes involved
in protein refolding and steroid receptor hormone binding.
Hsp90 Mutants--
Residues for mutation in hsp90 were selected
by a review of E. coli DNA gyrase B residues involved in ATP
binding and hydrolysis (38, 39). This revealed three residues shown to
participate in magnesium binding (Asn46, Ref. 39), adenine
ring interaction (Asp73, Ref. 39), and ATP hydrolysis
(Glu42, Ref. 38). Corresponding residues in hsp90 proteins
were determined by examination of published sequence comparisons
between hsp90 and gyrase B (40, 41). The particular residues in chicken hsp90 Protein Purification--
Purification of hsp70, Hop, ydj-1, and
p23 were performed as described previously (9, 11). For wild type and
mutant hsp90 proteins, bacterial pellets containing
overexpressed hsp90 were lysed by sonication in 10 mM
Tris-HCl, 10 mM EDTA, 10 mM thioglycerol, pH
7.5, containing a protease inhibitor mixture of 0.1 mM
leupeptin, 0.1 mg/ml bacitracin, 77 µg/ml aprotinin, 1.5 µM pepstatin, and 1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride. The soluble lysate fraction
was separated by sequential DEAE-cellulose and heparin-agarose column
chromatography, followed by Mono Q FPLC (Pharmacia) as described
previously (22). Purified protein was dialyzed and stored in either 10 mM Tris-HCl, 50 mM KCl, 0.1 mM
EDTA, 1 mM dithiothreitol, 10% glycerol, pH 7.5, or 10 mM Tris-HCl, 50 mM KCl, 5 mM
MgCl2, 2 mM dithiothreitol, pH 7.5. Protein
concentration was determined by scanning densitometric analysis of
Coomassie-stained SDS-PAGE gels containing known standards of hsp90 as
determined by amino acid analysis.
ATP-Sepharose Binding--
ATP-Sepharose binding was typically
performed with 5 µg of purified hsp90 in a final volume of 200 µl
of incubation buffer (IB: 10 mM Tris-HCl, 50 mM
KCl, 5 mM MgCl2, 2 mM
dithiothreitol, pH 7.5) containing 0.01% Nonidet P-40 (Sigma,
currently available as IGEPAL CA-630), and 20 mM
Na2MoO4, with or without 5 mM ATP. To this mixture was added 25 µl of p23 Binding--
p23 binding assays were typically performed in
a final volume of 200 µl of IB (see above) containing 0.01% Nonidet
P-40, 20 mM Na2MoO4, 5 mM ATP, and an ATP regeneration system consisting of 10 mM phosphocreatine and 7 units of creatine phosphokinase (both Sigma). Variations in nucleotide content are described for each
experiment in the figure legends. Five-10 µg of purified p23 were
used along with either 5 µg of purified hsp90 or 50 µl of cell
lysate from E. coli BL21 strains overexpressing hsp90. In
cases where cell lysate was used, expression of wild type and mutant
hsp90s was similar (data not shown). This mixture was incubated for 30 min at 30 °C. After incubation, the reactions were chilled on ice
and added to approximately 20 µg of anti-p23 monoclonal antibody JJ3
(45) bound to 25 µl of protein A-Sepharose (Pharmacia) for a 1-h
immunoprecipitation on ice with frequent mixing. The resin pellets were
then washed with 4 × 1 ml of cold IB. Bound proteins were
visualized by SDS-PAGE as described above.
Hop Binding--
Hop (p60) binding assays were performed in a
final volume of 200 µl of IB (see above) supplemented with 0.01%
Nonidet P-40 and 20 mM Na2MoO4,.
Each sample contained 5 µg of purified hsp90. In addition, some
samples also contained 5 mM ATP with or without 5 µg of
purified p23 as indicated. This mixture was incubated for 30 min at
30 °C. Following incubation, samples were chilled on ice and 5 µg
of purified Hop was added, along with 25 µl of protein A-Sepharose
containing 10 µl of F5 antibody against Hop (46). Hop was then
immunoprecipitated on ice for 1 h with frequent mixing. Next, the
antibody resin pellets were washed with 4 × 1 ml of cold IB.
Bound proteins were analyzed by SDS-PAGE as described above, and the
amount of hsp90 co-precipitating with Hop was quantified by scanning
densitometry of the Coomassie Blue-stained gel.
Phenyl-Sepharose Binding--
In the phenyl-Sepharose binding
assay, 5 µg of hsp90 were incubated at 30 °C for 20 min in a final
volume of 200 µl of IB (see above) containing 0.01% Nonidet P-40 and
20 mM Na2MoO4; with or without 5 mM ATP and the ATP regeneration system described above.
Following this incubation, the mixtures were chilled on ice, then added
to 25 µl of phenyl-Sepharose (Sigma) for a second incubation for 15 min on ice with frequent mixing to keep the resin in suspension. The
resin was then pelleted, and non-binding hsp90 in the supernatant was
removed. Phenyl-Sepharose pellets were washed with 3 × 1 ml of
cold IB containing 0.01% Nonidet P-40 and 20 mM
Na2MoO4. Proteins still bound to the resin were analyzed by SDS-PAGE as described above.
Firefly Luciferase Refolding--
Luciferase refolding assays
were performed as described previously (9, 12). Firefly luciferase
(Sigma) at 100 nM in stability buffer (25 mM
Tricine-HCl, 8 mM MgSO4, 0.1 mM
EDTA, 10 mg/ml bovine serum albumin, 10% glycerol, 0.25% Triton
X-100, pH 7.8) was denatured at 40 °C for 15 min to <1% of its
original activity. It was then diluted 1:10 in Tris buffer (TB: 10 mM Tris-HCl, 3 mM MgCl2, 50 mM KCl, 2 mM dithiothreitol, pH 7.5) containing
2 mM ATP, an ATP-regenerating system (10 mM
phosphocreatine and 3.5 units/100 µl of creatine kinase), and
purified chaperone proteins, in a final volume of 35 µl. In addition,
some tubes contained the hsp90 inhibitor geldanamycin (Drug Synthesis
and Chemistry Branch, Developmental Therapeutics Program, Division of
Cancer Treatment, NCI, National Institutes of Health), at a
concentration of 18 µM. This mixture was incubated at
25 °C to promote refolding, which was assayed at each time point by
measuring the luciferase activity of a 5-µl aliquot in a luminometer
(Turner). The chaperone concentrations used were: 550 nM
hsp70, 160 nM ydj-1, 100 nM Hop, and 180 nM hsp90 (calculated as a monomer). Under these conditions,
the effect of hsp90 was unchanged by varying its concentration within
the range 100-600 nM (data not shown).
Progesterone Receptor Complex Assembly--
Chicken oviduct
cytosol preparation, progesterone receptor (PR) isolation, and PR
complex assembly were largely performed as described previously (8),
and they will only be described in brief here. Chicken oviduct cytosols
were prepared at 4 °C by homogenization of frozen oviducts from
estrogen-treated chickens in 4 volumes of homogenization buffer (10 mM Tris-HCl, 10 mM EDTA, 10 mM
thioglycerol, pH 7.5, containing a protease inhibitor mixture of 0.1 mM leupeptin, 0.1 mg/ml bacitracin, 77 µg/ml aprotinin, 1.5 µM pepsatatin, and 1 mM AEBSF
(4-(2-aminoethyl) benzenesulfonyl fluoride)). Lipid and insoluble
material were removed by centrifugation (10 min at 16,000 × g, followed by 1 h at 110,000 × g,
keeping the supernatant and avoiding the lipid layer each time).
Cytosol was treated at 4 °C with 0.5 M KCl and 5 mM ATP for 30 min to strip PR of all coprecipitating
proteins. Monoclonal antibody PR22 (47) was conjugated to protein
A-Sepharose resin (Pharmacia) for use in PR isolation, using 1 mg of
antibody/1 ml of resin. Treated cytosol (1.6 ml) was added to 25 µl
of antibody resin, and PR was adsorbed by a 1.5-h incubation at 4 °C
with constant, gentle mixing. Following adsorption, pellets were washed
with 3 × 1 ml homogenization buffer and once with 1 ml of IB (see
above) to remove unbound proteins.
PR complexes were formed by incubation of PR pellets with 20 µg of
hsp70, 20 µg of hsp90, 5 µg of Hop, 2 µg of ydj-1, and 5 µg of
p23 in a final volume of 200 µl of IB containing 5 mM ATP
and 0.01% Nonidet P-40 for 30 min at 30 °C with frequent mixing. Where indicated, geldanamycin was added at a concentration of 10 µg/ml (18 µM). Following incubation, the mixtures were
chilled on ice and supplemented with a mixture of 5 nM
[1,2-3H]progesterone (1.9 TBq/mmol, NEN Life Science
Products Inc.) and 100 nM unlabeled progesterone. Samples
were then incubated for 1 h at 4 °C with constant mixing to
allow progesterone binding to the receptor. Unbound hormone was washed
away with 3 × 1 ml of cold IB. The antibody resin was resuspended
in 1 ml of IB, and 100 µl was removed for scintillation counting of
bound [3H]progesterone. The remaining resin was pelleted
and analyzed by SDS-PAGE as described above.
The N50A and D92A Mutations Prevent Hsp90 Binding to
Magnesium ions are necessary for ATP binding to hsp90 (22, 25),
providing a possible point for disruption of binding. An asparagine
residue (corresponding to Asn50 in chicken hsp90
The effect of these mutations on hsp90 binding to ATP-Sepharose is
shown in Fig. 1. In addition to wild type
hsp90, only the E46A mutant was capable of binding to the resin.
However, it appears that E46A does not bind as well as does wild type
hsp90 (compare protein loads to bound protein for each). The inclusion
of 5 mM free ATP effectively competes away binding by both
the wild type and E46A. In contrast, neither N50A nor D92A is capable
of binding immobilized ATP. This is shown by the small amount of bound
protein and the absence of an effect of competing free ATP, indicating that binding is nonspecific. Thus, while mutation of Glu46
has been shown to disrupt hydrolysis of ATP in comparable systems (36,
37), it is only partially effective in preventing binding to ATP. This
provides us with an ATP-binding hsp90 that should be deficient in
hydrolysis for use in our study. The inability of hsp90 with mutations
at Asn50 or Asp92 to bind ATP-Sepharose
demonstrates that interactions between hsp90 and complexed
Mg2+ and adenine, respectively, are essential for binding.
These two mutants, then, are used to show the effect of the loss of ATP binding on hsp90 function. Note that these bacterially expressed hsp90
proteins are not as pure as the baculovirus-expressed ones used in our
earlier work (22), which is due to comparatively low expression
relative to endogenous bacterial proteins. However, these co-purifying
proteins do not appear to interfere with the assays performed here,
since side by side controls using baculovirus-expressed wild type hsp90
showed results similar to those using its bacterially expressed
counterpart (data not shown). The identity of the contaminants is not
known, although the largest molecular weight contaminant in the wild
type hsp90 preparation appears to be a degradation product of hsp90
that is able to bind ATP-Sepharose (Fig. 1).
Hsp90 Binding to p23 Does Not Require ATP Hydrolysis--
The
co-chaperone p23 was first identified as a component of unactivated
progesterone receptor complexes containing hsp90 (50). At the time of
its discovery, it was unclear what role p23 was playing in the
maturation of steroid receptors to their steroid-binding conformations.
Isolated PR incubated in reticulocyte lysate complexed with hsp90 only
in the presence of p23 (51). However, more recent studies of
glucocorticoid receptor complex formation from purified components have
shown that p23 is not required to fold glucocorticoid receptor into its
steroid-binding conformation (52). Instead, it promotes the persistence
of this conformation, thereby increasing the efficiency of the process
(7).
Complexes consisting only of hsp90·p23 have been formed using
purified proteins in a process requiring ATP and promoted by molybdate
ions (11). The role of ATP appears to be to switch hsp90 from a
conformation with low affinity for p23 to one with high affinity (22).
The role of molybdate ions is less clear. Molybdate, as well as
vanadate and tungstate, have been known for many years to stabilize
steroid receptors in an unactivated, steroid-binding state (5), and
this stabilization occurs through the maintenance of hsp90 binding to
the receptor (53, 54). Even more effective than ATP plus molybdate,
however, is the poorly hydrolyzable nucleotide analog, ATP
A comparison of the ability of wild type, E46A, N50A, and D92A hsp90
forms to bind to p23 is shown in Fig. 2.
Lysate from E. coli strains overexpressing hsp90 was
incubated at 30 °C with purified p23 in the presence or absence of
ATP and an ATP regenerating system. Following incubation, p23 was
immunoprecipitated, and proteins co-precipitating with p23 were
visualized by SDS-PAGE. The absolute requirement for ATP in generating
hsp90·p23 complexes is seen by the absence of significant complex
formation when ATP is not present. This prerequisite is seen again with
the ATP binding-deficient mutants, N50A and D92A, which are unable to
bind p23 regardless of the presence of ATP. Wild type hsp90 and the
hydrolysis-deficient mutant, E46A, on the other hand, are both able to
complex p23 when ATP is provided. This confirms that hsp90 needs only
to bind ATP to undergo the conformational switch to a form with high
affinity for p23.
The Role of Molybdate in Hsp90 Function Is to Stabilize the
ATP-bound State after Hydrolysis--
The above results with wild type
and E46A hsp90 encouraged us to further analyze the nucleotide
requirements for p23 binding. We compared the effectiveness of ATP, ATP
plus molybdate, and ATP The High ATP Concentrations Required to Affect Hsp90 Are at Least
Partially Due to Hydrolysis--
In order to demonstrate the effect of
ATP on hsp90 in vitro functions, such as the binding of p23,
it has been necessary to use high concentrations of ATP, typically 5 mM (9, 11, 22). The results above show that the improved
efficacy of ATP Hsp90 Has Reduced Affinity for Hop Upon ATP Binding, but Hydrolysis
Is Not Necessary to Effect this Change--
Hop (hsp
organizing protein, also known as p60) is a
co-chaperone known to associate with both hsp90 and hsp70 (46). Cell lysates from several tissues show complexes of Hop with hsp70 and hsp90
(46), and Hop has also been shown to form complexes with each
individual hsp in vitro (9, 55). Because hsp70 and hsp90 do
not bind each other (55), Hop may serve as a mediator to bring these
two heat shock proteins together. The importance of this association is
seen in steroid receptor complexes, where Hop is essential for
obtaining the steroid-binding conformations of both glucocorticoid and
progesterone receptors in vitro (8, 56). Hop also
accelerates refolding of denatured firefly luciferase by hsp70 and
hsp40 when hsp90 is present (9). Hsp90 and hsp70 appear to bind to
separate domains of Hop, allowing the trimeric complex to form
(55).
Hop binding by the hsp70 and hsp90 chaperones is regulated by
nucleotides. Both hsp70 and hsp90 favor Hop binding in their ADP-bound
conformations, and for hsp90, once Hop is bound, this conformation is
stabilized (9). Similarly, stabilization of the ATP-bound conformation
of hsp90 by p23 inhibits Hop binding (9). Here, we sought to further
analyze the mechanism of these conformational changes of hsp90 with the
use of the E46A and N50A mutants. Wild type or mutant hsp90 was
preincubated under conditions favoring either the ADP-bound or
ATP-bound conformation. The ADP conformation of hsp90 was generated by
preincubation in the absence of nucleotides, since our earlier work has
shown that hsp90's default conformation without nucleotide is very
similar to its conformation with ADP (11). The ATP conformation was
generated using ATP, with or without p23. Following preincubation, Hop
was added and then immunoprecipitated. The amount of hsp90
co-precipitating with Hop was measured, and the results are shown in
Fig. 4.
With wild type hsp90, only a small reduction in Hop binding is seen
with ATP addition. However, p23 stabilization of this conformation
reduces Hop binding to 60% of levels seen with no nucleotide present.
E46A, on the other hand, requires only ATP to achieve a reduction in
Hop binding to 60%, and the addition of p23 does not further reduce
binding. Continued binding of the E46A mutant to Hop in the presence of
ATP may indicate that the reduction in affinity for Hop is not great
enough to prevent binding under these conditions. It may also reflect
E46A's lower affinity for ATP (Fig. 1), whereby it spends less time
with ATP bound and more time in a Hop-binding conformation. Unable to
bind ATP, the N50A mutant binds Hop similarly in the presence or
absence of ATP, and p23 does not potentiate a reduction in Hop binding.
These results indicate that the conformation of hsp90 with low affinity for Hop is brought about by ATP binding alone and that hydrolysis is
unnecessary. Indeed, hydrolysis appears to return hsp90 to its
high-affinity state for Hop. p23's role in stabilization of the
low-affinity state is to bind to hsp90 while it holds ATP, keeping it
in this conformation after hydrolysis has occurred or preventing hydrolysis.
ATP Binding, but Not Hydrolysis, Is Necessary to Reduce Hsp90
Affinity for Phenyl-Sepharose--
The hydrophobic resin,
phenyl-Sepharose, has been suggested as a model representing a
hydrophobic protein substrate for hsp90 (21). Hsp90 binding to this
resin does not require ATP (11), supporting the use of the model, since
hsp90 binding to selected substrate proteins in vitro has
also been ATP independent (13, 16, 19, 57, 58). For one of these
substrates, MyoD, it has been shown that the site of binding on hsp90
lies in the carboxyl portion of the protein (58). Similarly, our
studies with hsp90 fragments have shown that interaction with
phenyl-Sepharose occurs outside of the amino-terminal ATP-binding
domain.2 However, if hsp90 is
preincubated with ATP, it will show reduced binding to
phenyl-Sepharose, while ADP preincubation does not have an effect (11).
It is not clear if ATP hydrolysis is necessary for hsp90 to adopt a
conformation which shows reduced affinity for phenyl-Sepharose. To
answer this question and to confirm the need for ATP to effect a
structural change in hsp90, we have tested the E46A and N50A mutants
along with wild type hsp90 in this assay, the results of which are
shown in Fig. 5.
Hsp90 was preincubated at 30 °C in the presence or absence of ATP
and an ATP regeneration system. Following this preincubation, the
mixtures were chilled on ice, and phenyl-Sepharose resin was added for
a second incubation on ice to allow hsp90 to bind. The resin pellets
were washed, and the remaining protein bound was examined. The presence
of E46A and N50A mutations in the ATP-binding domain do not prevent
hsp90's binding to phenyl-Sepharose in the absence of nucleotide, as
both appear to bind as well as wild type. However, the addition of ATP
to the N50A mutant has no effect on the amount of hsp90 bound, while
wild type and E46A forms show a marked decrease in binding. Thus, loss
of ATP binding prevents the conformational change necessary to reduce
hsp90's affinity for phenyl-Sepharose. The ATP effect seen with E46A
indicates that although ATP binding is necessary for this change,
hydrolysis is not.
ATP Binding and Hydrolysis Are Necessary for Hsp90 to Fully Assist
Hsp70 in Refolding Thermally Denatured Firefly Luciferase--
Hsp70
and ydj-1 (a member of the hsp40 family) have been shown in
vitro to form a protein folding system capable of refolding thermally denatured firefly luciferase (12). Although hsp90 is not an
essential component of this system, it appears to have a stimulatory
effect on the process (9, 12). Moreover, the addition of the
co-chaperone Hop generates a synergy whereby the combined effect of Hop
and hsp90 is greater than the sum of their individual effects (9). The
beneficial effect of hsp90 on luciferase refolding is only partially
reduced by geldanamycin, an hsp90-specific inhibitor which binds to the
same site on hsp90 as does ATP (22, 25, 26), implying there are
separate ATP-dependent and ATP-independent functions of
hsp90 in this process (12).
To better understand the role of ATP in hsp90-mediated protein folding,
the ATPase-deficient mutant E46A and the ATP binding-deficient mutant
N50A were compared with wild type hsp90 in a refolding system including
hsp70, ydj-1, and Hop. In this assay, firefly luciferase was thermally
denatured at 40 °C, and activity was confirmed to be <1% of its
original activity. It was then added to a mixture containing ATP, an
ATP-regenerating system, hsp70, ydj-1, and Hop, with or without hsp90,
and allowed to refold at 25 °C. To specifically remove the
ATP-dependent function of hsp90, some reactions also
included the inhibitor geldanamycin (GA). Refolding was measured by the
return of luciferase activity, shown by the results from a typical
experiment in Fig. 6A. As
demonstrated by the accelerated rise in luciferase activity, all three
hsp90 forms tested had a positive effect on refolding in the presence of Hop. This effect is not the result of simply increasing total protein concentration of the reaction, as the mixture already contains
over 60-fold more bovine serum albumin than it does hsp90. The success
of mutant hsp90s in promoting refolding indicates that hsp90 has some
ATP-independent chaperoning function.
Fig. 6B shows the fold stimulation of luciferase activity
over hsp70/ydj-1/Hop alone. Wild type hsp90 provided the most benefit by far (p < 0.05 by two-way ANOVA over the time course
studied, with a comparison of five experiments), with its greatest
impact early in the refolding process. As was seen in vivo
in yeast (59), hsp90 speeds refolding without significantly changing
the final level of luciferase activity. This is shown by the decreasing effect of hsp90 at the later time points. The N50A mutant was consistently superior to E46A in the absence of GA, although this difference was not statistically significant (p = 0.06 by two-way ANOVA, with a comparison of six experiments). Interestingly,
the addition of GA produced similar refolding curves in all three hsp90
forms (Fig. 6, A and B). In order to produce such
a result, GA must have a different effect on each hsp90 tested. When
the differences in activity caused by GA addition are assessed (Fig. 6C), wild type hsp90 loses up to 40% of its activity with
GA, while E46A increases activity by about 10-15% and N50A is
unaffected. The differences in GA effect on each mutant are
statistically significant over the time course studied
(p < 0.05 by two-way ANOVA). These results show that
ATP binding and hydrolysis are essential for optimal hsp90
participation in refolding with hsp70. Moreover, E46A's ability to
bind ATP without hydrolysis places hsp90 in a state with inhibitory
properties relative to that of an hsp90 molecule unable to bind ATP at
all, and this inhibition is relieved by GA. Finally, the absence of a
GA effect on the N50A mutant indicates that GA acts only by
displacement of ATP.
Hsp90 Must Hydrolyze ATP to Fold the Progesterone Receptor into a
Steroid Binding Conformation--
Molybdate ions have been known to
stabilize the mature (i.e. steroid-binding) form of the
progesterone receptor from chicken oviduct cytosols (60, 61). This form
of the receptor is not alone, but in a complex including hsp90, p23,
and an immunophilin (FKBP52, FKBP51, or Cyp40) (62). It is believed
that in the process of forming this complex, hsp90 forms an earlier,
intermediate complex with the receptor that contains hsp70 and Hop
(63). Recently, techniques have been developed for assembling
steroid-binding receptor complexes from purified proteins. For the
glucocorticoid receptor, a series of papers by Dittmar et
al. (6, 7, 52) has shown that this requires the addition of hsp70,
hsp40, hsp90, Hop, and, for stabilization, p23. Our laboratory has
assembled PR complexes with the same set of proteins (8). Although ATP is essential for assembly, it is not clear where in the process hsp90
utilizes ATP, if at all, and whether or not this utilization involves
hydrolysis. Hsp90·p23 complex formation from purified proteins
requires ATP, but poorly hydrolyzed ATP
We immunoprecipitated PR from chicken oviduct cytosol and stripped it
of bound proteins. Using purified hsp70, ydj-1, Hop, hsp90, and p23, we
reassembled PR complexes at 30 °C in the presence of ATP and
molybdate. As shown in Fig.
7A, hsp90 does not have to
bind ATP in order to form a complex with PR. The amount of hsp90
present in all cases, including in the presence of the inhibitor GA and
the N50A mutation, is greater than the nonspecific background binding
seen when PR is not included, although the wild type without GA
provides the greatest amount of hsp90 co-precipitating with PR.
Similarly, loss of ATP hydrolysis does not exclude complex formation,
as indicated by the binding seen with the E46A mutant. The type of
complex formed, however, is affected by the ability to bind and
hydrolyze ATP. Only by using wild type hsp90 in the absence of
geldanamycin will the mature, p23-containing complex be created. The
other complexes seen all appear to be of the intermediate type,
containing hsp70, Hop, and hsp90 (as well as ydj-1). For wild type
hsp90, GA inhibition changes the complex formed from the mature type to
the intermediate complex, indicated best by the presence or absence of
p23. On the other hand, E46A and N50A form this complex regardless of
the presence of GA. Thus, the mutants are "self-inhibitory" in that
they cannot progress beyond the intermediate complex without ATP
binding and hydrolysis.
Each of these complexes was then incubated at 4 °C with radiolabeled
progesterone to assess its ability to bind steroid, indicating the
adoption of the PR hormone binding conformation, and the corresponding levels of progesterone binding are shown in Fig. 7B. Not
surprisingly, wild type hsp90 best supports the steroid-binding
conformation of the PR, with most of this activity lost in the presence
of GA. The level of hormone binding does not simply correlate with the
amount of hsp90 present, since the dramatic loss of progesterone binding seen with the E46A mutant is accompanied by a much smaller decrease in the amount of E46A protein complexing with receptor (Fig.
7A). The mutant hsp90s, without p23 in their complexes, cannot properly fold PR to allow hormone binding. The effect of GA on
them is minimal, since the loss of ATP binding or hydrolysis is already
sufficient to lock their receptors in a non-progesterone-binding state.
Under these conditions, the presence of a p23-containing complex
appears to be more important than binding of hsp90 in the generation of
a hormone binding conformation of PR. The assembly of this complex
requires ATP binding as well as hydrolysis by hsp90. The absolute
requirement for hsp90 in stabilizing the hormone-binding state is shown
by the absence of progesterone binding when hsp90 is not included
during incubation.
With recent evidence that hsp90 not only binds ATP (22-25), but
also hydrolyzes it (18, 36, 37), it is important to examine how the
functions of hsp90 are affected by these processes. At least some key
functions of hsp90 that are required for yeast viability are dependent
upon its ability to bind and hydrolyze ATP (36, 37). Using ATP binding
and hydrolysis mutants, we have been able to separate the individual
effects of different steps of hsp90's ATP cycle. Our studies here
indicate that in vitro associations between hsp90 and p23,
Hop, and phenyl-Sepharose are regulated only by the conformational
changes induced by binding of ATP. This is evidenced by
hydrolysis-deficient mutant E46A's ability to respond to ATP as well
as, or better than, wild type hsp90 in these three assays. It is
interesting that ATP binding supports binding to p23, yet reduces
affinity for Hop and phenyl-Sepharose. Thus, hsp90 appears to have
unique sets of associated proteins and substrates for each nucleotide
state. The different PR complexes reconstituted from reticulocyte
lysate depending on the inclusion of GA (which blocks the ATP-bound
state of hsp90) seem to support this idea (62). ATP appears to be
readily hydrolyzed under the conditions used here. The significance of
hydrolysis in p23, Hop, and phenyl-Sepharose binding assays appears to
be to simply reset hsp90 to an ADP-bound or a nucleotide-free
conformation, thereby making the ATP-bound conformation transient. This
explains the enhanced effectiveness of ATP The functions of ATP binding and hydrolysis were tested in two
multiprotein complex-based systems: the folding of luciferase and the
assembly of PR complexes. These systems differ in their requirement for
hsp90. While hsp90 is essential for chaperoning PR to its hormone
binding state, it is not essential for luciferase folding, although it
stimulates this process. However, ATP binding and hydrolysis are both
required for optimal effects on luciferase folding and for PR
chaperoning, perhaps because both of these processes require the
dynamic assembly and disassembly of complexes (63). Unlike what is seen
in the formation of the binary complexes described above, ATP binding
alone is not sufficient for maximum refolding efficiency. Indeed, ATP
binding without hydrolysis appears to make hsp90 less effective in the
luciferase assay than if it were not bound at all.
Our results show that hsp90 has an ATP-independent dimension to its
activity. ATP-negative mutants can enhance the in vitro folding of luciferase, although suboptimally, and they can enter PR
complexes, but are unable to chaperone the PR to a hormone binding
state. These results indicate that hsp90 can bind and release substrate
proteins passively and are consistent with earlier studies showing that
hsp90 passively maintains unfolded proteins in a non-aggregated state
capable of folding (12, 13, 15-19). However, the full activity of
hsp90 may be significantly more complex. In the PR system, hsp90
appears to accept the substrate from hsp70 through a process that is
facilitated by Hop (8, 55). The dynamics of this process are unknown,
but in vitro, it takes several minutes to accomplish. This
suggests that the transfer of PR from hsp70 to hsp90 is not simple and
may involve time-consuming events such as major conformational changes
of chaperones and substrate. It is also possible that this is an iterative process where the substrate is passed back and forth between
hsp70 and hsp90 until it assumes a more advanced folding state. Proper
coordination of substrate binding and release may only occur when hsp90
can both bind and hydrolyze ATP.
In the luciferase folding system, protein-protein interactions and
complexes have not yet been well defined. However, it is likely that
this chaperoning process also involves a dynamic hsp70·Hop·hsp90 complex similar to that for PR since the system functions best when all
three proteins are present. In addition, this system may be facilitated
by passive interactions between denatured luciferase and hsp90 as
demonstrated by previous studies which have shown that hsp90 can, to a
lesser extent, enhance luciferase folding in the absence of Hop (9,
12).
Although p23 does not appear to have a role in the luciferase folding
system,3 it is clearly
important for PR complexes. The binding of hsp90 to p23 requires ATP
binding, but not hydrolysis. In contrast, the PR chaperoning system
needs both ATP binding and hydrolysis. This suggests that p23 binding
is influenced by substrate binding or other proteins in the PR system.
It would seem that hsp90 can assume a conformation capable of p23
binding only after the substrate (PR) has progressed to a near-native
conformational state, which requires ATP hydrolysis as described above.
Indeed, studies by Pratt and co-workers (7) suggest that p23 can bind
hsp90 in the absence of added ATP when hsp90 is bound to the
glucocorticoid receptor in an advanced stage of folding. Thus, there
may be multiple factors that can influence the conformational state of hsp90.
In considering the interaction of substrates with hsp90, one needs to
consider the possibility that hsp90 has two substrate-binding sites.
Both Young et al. (19) and Scheibel et al. (18)
have shown that the passive chaperoning activity of hsp90 can be
accomplished by fragments of the protein near the NH2
terminus and also, near the COOH terminus. Thus, protein substrates may
be transferred from one binding site to the other. If so, this process
may be controlled by ATP binding or hydrolysis.
Another potential complexity can be envisioned by comparing hsp90 to
topoisomerase II, which functions as a dimer and contains an
ATP-binding domain with similarities to hsp90 (26, 36). Recent studies
indicate a complex, sequential pathway for ATP binding and hydrolysis
by topoisomerase II which probably relates to multiple conformational
states of the protein (65, 66).
The present studies illustrate the roles of ATP binding and hydrolysis
in various hsp90/protein interactions. They provide a basis for future
studies that are needed to describe more clearly the characteristics of
ATP binding and hydrolysis by hsp90 and the resulting conformational
and functional states of this protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
were found using a published analysis of hsp90 genes from a
wide range of species (1). The equivalent residues between E. coli gyrase B and chicken hsp90
are, respectively:
Asn46 and Asn50; Asp73 and
Asp92; Glu42 and Glu46. These were
each mutated individually to alanine residues through the use of
polymerase chain reaction-directed overlap extension as described
previously (42) using clone p7.11 containing the coding sequence for
chicken hsp90
(22) as the template. These mutant genes were then
subcloned into the pET23 vector (Novagen) for overexpression in
E. coli BL21(DE3)pLysS cells (Novagen) following the
manufacturer's protocols.
-phosphate-linked ATP-Sepharose resin (provided by Timothy Haystead (Ref. 43), commercially available
from Upstate Biotechnologies, Inc.) which was washed and
pre-equilibrated with IB before use to remove free nucleotides. Samples
were then incubated for 20 min at 30 °C with frequent mixing to
resuspend the resin. After incubation, samples were chilled on ice and
the resin was pelleted. Following removal of unbound protein in the
supernatant, resin pellets were washed with 3 × 1 ml of cold IB.
Bound proteins were eluted from the resin by boiling 2 min in SDS
sample buffer (2% SDS, 5%
-mercaptoethanol) and resolved by
SDS-PAGE (44).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Phosphate-linked ATP-Sepharose, while E46A Does Not--
Protein
sequence analyses of type II DNA topoisomerases and the MutL family of
DNA repair enzymes revealed the presence of three regions in hsp90's
amino-terminal domain containing sequence homology to these proteins
(40, 41). In addition, a predicted structure for this domain of hsp90
suggested similarities with the NH2-terminal domain of the
B subunit of DNA gyrase (48). This domain of gyrase B is known to
contain the ATP binding and hydrolysis functions of the subunit (49),
and its crystal structure has been determined (39). We used studies of
gyrase B's structure and function along with structural and sequence
data for hsp90 to find residues in hsp90 which could be involved in ATP
binding and hydrolysis.
)
interacts with a magnesium ion in the gyrase B crystal structure (39),
and the same interaction is believed to be found in hsp90 (25). We
changed this residue to alanine, and the corresponding mutant is called
N50A. The second residue we altered was an aspartate at position 92. The crystal structures for gyrase B and hsp90 show that this residue
hydrogen bonds with the adenine base (25, 39) of ATP. The likelihood of
its importance is enhanced by the fact that it is a charged residue in
an otherwise hydrophobic environment (25, 26). Asp92 was
changed to an alanine, producing the D92A mutant. Finally, we sought to
generate a mutant hsp90 which would bind ATP, but be unable to
hydrolyze it. A study of the gyrase B ATPase reaction demonstrated that
mutation of a glutamate residue (corresponding to Glu46 in
chicken hsp90
) to alanine or glutamine eliminated ATPase activity
while still allowing ATP binding (38). We chose to mutate this residue
to alanine, resulting in the E46A mutant. A phylogenetic study of hsp90
proteins shows that the three residues we have selected for mutation
are conserved from E. coli to humans (1), further indicating
their importance. Two of the residues described here have recently been
mutated in yeast hsp90 (36, 37) and human hsp90 (37). Those studies
demonstrated that changing Asp92 (Asp79 in
yeast) to asparagine reduces ATP binding to subdetectable levels, and
that changing Glu46 (Glu33 in yeast) to alanine
reduces ATPase activity to <1% of wild type while keeping ATP binding
comparable to wild type.
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Fig. 1.
ATP-Sepharose binding by wild type and mutant
hsp90 proteins. 5 µg of hsp90 was incubated for 20 min at
30 °C with 25 µl of -phosphate-linked ATP-Sepharose in a final
volume of 200 µl of incubation buffer (see "Experimental
Procedures") supplemented with 20 mM molybdate and 0.01%
Nonidet P-40. Free ATP at a concentration of 5 mM was
included (+) or left out (
) as indicated. After
incubation, the resin was washed and bound hsp90 was eluted and
analyzed by SDS-PAGE. 5 µg of protein loads of each hsp90 are shown
on the right.
S, which
is not affected by molybdate (11), indicating that the role of
molybdate is linked to ATP hydrolysis. Thus it appears that the
mechanism of molybdate enhancement of hsp90·p23 complex formation is
that molybdate binds in the hsp90 nucleotide pocket upon hydrolysis of
ATP, keeping hsp90 in its ATP-bound conformation (11). The
effectiveness of ATP
S implies that hydrolysis, although it occurs,
is not necessary for p23 binding by hsp90. Here, through the use of ATP
binding- and hydrolysis-deficient mutants, we can directly study the
importance of these functions in hsp90·p23 complex formation, as well
as test our explanation for molybdate's effect.
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Fig. 2.
p23 binding by wild type and mutant hsp90
proteins. 10 µg of p23 and 50 µl of lysate from E. coli strains overexpressing hsp90 were incubated for 30 min at
30 °C in a final volume of 200 µl of incubation buffer (see
"Experimental Procedures") supplemented with 20 mM
molybdate and 0.01% Nonidet P-40, in the presence (+) or
absence ( ) of 5 mM ATP and an ATP regeneration system.
Following incubation, p23 was immunoprecipitated, and bound proteins
were eluted and analyzed by SDS-PAGE. Bands corresponding to hsp90,
p23, and anti-p23 antibody heavy (HC) and light
(LC) chains are indicated.
S in supporting p23 binding by each hsp90
(Fig. 3A). In the absence of
molybdate, ATP is not very effective in promoting p23 binding by wild
type hsp90. p23 binding is improved when molybdate is added, or if
ATP
S is used instead of ATP. In contrast, the E46A mutant binds p23
well, regardless of the nucleotide used. These results indicate that
the benefit of molybdate arises from its ability to stabilize an
ATP-bound conformation of hsp90 only after ATP has been hydrolyzed. If
ATP is not being hydrolyzed, as with E46A or when ATP
S is used,
molybdate is not required. Although hsp90's ATPase activity has been
reported as low when compared with other ATP-hydrolyzing enzymes (18,
24, 36, 37), ATP is apparently being hydrolyzed under the conditions used here.
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Fig. 3.
Nucleotide requirements for p23 binding by
wild type and E46A mutant hsp90. A, 10 µg of p23 and
5 µg of hsp90 were incubated for 30 min at 30 °C in a final volume
of 200 µl of incubation buffer (see "Experimental Procedures")
supplemented with 0.01% Nonidet P-40, along with either 2 mM ATP or 1 mM ATP S as indicated, in the
presence (+) or absence (
) of 20 mM molybdate
(MoO4
). Following incubation, p23 was
immunoprecipitated, and bound proteins were eluted and analyzed by
SDS-PAGE. Bands corresponding to hsp90, p23, and antibody heavy
(HC) and light (LC) chains are indicated.
B, 5 µg of p23 and 5 µg of hsp90 were incubated for 30 min at 30 °C in a final volume of 200 µl of incubation buffer
supplemented with 0.01% Nonidet P-40 and ATP at the concentrations
shown, along with an ATP regeneration system. Following incubation, p23
was immunoprecipitated, and bound proteins were eluted and analyzed by
SDS-PAGE. The band of hsp90 co-precipitating with p23 is shown.
S over ATP is lost when hydrolysis is not taking
place. Using the E46A mutant, we tested whether in the absence of
hydrolysis, a much lower concentration of ATP could still support p23
binding. The results in Fig. 3B demonstrate that in the
absence of molybdate, a 100-fold lower concentration of ATP is
sufficient to give the same p23 binding to E46A as compared with wild
type hsp90 (compare wild type binding at 5 mM with E46A
binding at 50 µM). When one considers that E46A may have
reduced affinity for ATP (see Fig. 1), it is conceivable that wild type
hsp90 can be influenced by even lower concentrations of ATP. We have
previously demonstrated that lower nucleotide concentrations may be
used with ATP
S (11), but the concentrations used here are lower
still. Also, it is possible that the increased effectiveness of ATP
S
is due to a difference in hsp90's affinity for it. Thus, while the
formation of stable hsp90·p23 complexes in vitro may
require a high ATP concentration, our results suggest that a lower
concentration may still be sufficient to bring about
ATP-dependent changes in hsp90 activity.
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Fig. 4.
Hop binding by wild type and mutant hsp90
proteins. 5 µg of hsp90 was incubated for 30 min at 30 °C in
a final volume of 200 µl of incubation buffer (see "Experimental
Procedures") supplemented with 20 mM molybdate and 0.01%
Nonidet P-40; without ATP or p23 (black bars), with 5 mM ATP only (hatched bars), or with 5 mM ATP and 5 µg of p23 (white bars). Following
incubation, 5 µg of Hop was added and immediately immunoprecipitated
on ice. Bound proteins were eluted and analyzed by SDS-PAGE.
Co-precipitating hsp90 was quantified by densitometry of the gel.
Results are shown as percentages of the amount of hsp90 bound to Hop in
the absence of ATP and p23.
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Fig. 5.
Phenyl-Sepharose binding by wild type and
mutant hsp90 proteins. Five µg of hsp90 was preincubated for 20 min at 30 °C in a final volume of 200 µl of incubation buffer (see
"Experimental Procedures") supplemented with 20 mM
molybdate and 0.01% Nonidet P-40, with or without ATP and an ATP
regeneration system. The samples were then chilled on ice, and 25 µl
of phenyl-Sepharose was added. The samples underwent a second
incubation on ice for 15 min. After washing the resin, bound hsp90 was
visualized by SDS-PAGE. The band of hsp90 on the gel is shown. Lanes
show the 5 µg of hsp90 loaded onto the resin (Load), the
amount bound in the absence of ATP (Bound), and the amount
bound in the presence of ATP (+ATP).
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Fig. 6.
Luciferase refolding by hsp70, hsp40, Hop,
and wild type or mutant hsp90. 100 nM firefly
luciferase was thermally denatured at 40 °C to <1% of its original
activity. It was then diluted 1:10 in a final volume of 35 µl of Tris buffer (see
"Experimental Procedures") containing 2 mM ATP, an
ATP-regeneration system, 550 nM hsp70, 160 nM
ydj-1 (an hsp40 protein), and 100 nM Hop, with or without
180 nM hsp90 (calculated as a monomer). Wild type (w.t.
hsp90; squares), E46A (circles), or N50A
(triangles) hsp90 were used. This mixture was incubated at
25 °C, and refolding was assayed by measurement of luciferase
activity in a 5-µl aliquot removed at each time point. Some samples
also contained the hsp90 inhibitor GA at a concentration of 17.8 µM. A, typical refolding curves seen with each
form of hsp90 are shown. Refolding by hsp70, ydj-1, and Hop only
(No hsp90) is shown by the thick line
(diamonds). Samples containing GA are represented by
dashed lines and open symbols, versus
samples without GA, which are drawn with solid lines and
closed symbols. B, the fold increase in
luciferase activity with each hsp90 over the activity measured in the
absence of hsp90 is shown. This was calculated by dividing luciferase
activity at each time point in the presence of a given hsp90 by
activity in the absence of any hsp90. The inclusion of GA is indicated
as in A. These results are the means of five experiments.
C, the percent change in luciferase activity upon GA
addition is shown. This was calculated by comparing the activity at
each time point of a sample containing a particular hsp90 with that of
a sample treated identically except for the addition of GA. Results are
shown relative to the activity of the sample without GA, and they are
the means of six experiments.
S is superior to ATP in this
process (Fig. 3A and Ref. 11). On the other hand, hsp90-Hop
pairs form in the absence of ATP. Here, we use the E46A and N50A
mutants along with wild type hsp90 to assess the need for ATP binding
and/or hydrolysis by hsp90 in forming steroid-binding progesterone
receptor complexes.
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Fig. 7.
Assembly of PR complexes containing wild type
or mutant hsp90. Progesterone receptors were isolated from chicken
oviduct cytosol by immunoadsorption to antibody resin, and then
stripped of other proteins by treatment at 4 °C with 0.5 M KCl and 5 mM ATP. In order to regenerate
multiprotein complexes capable of binding hormone, purified hsp70,
ydj-1 (an hsp40 protein), Hop, p23, and wild-type (w.t.) or
mutant (E46A and N50A) hsp90 were added to the receptor resin in a
final volume of 200 µl of incubation buffer (see "Experimental
Procedures") with 5 mM ATP and 0.01% Nonidet P-40. Where
indicated (+GA), 17.8 µM geldanamycin was also
included. This mixture was incubated for 30 min at 30 °C.
Progesterone binding was assessed with the addition of a saturating
mixture of 3H-labeled and unlabeled progesterone followed
by incubation at 4 °C for 1 h. 1/10th of the PR resin was used
for scintillation counting of bound 3H-labeled
progesterone, and the remainder was boiled in SDS sample buffer for
evaluation of complexes by SDS-PAGE. A, SDS-PAGE of in
vitro assembled PR complexes is shown. Bands corresponding to the
A (PR-A) and B (PR-B) PR isoforms, hsp90, hsp70,
Hop, ydj-1, p23, and anti-PR antibody heavy (HC) and light
(LC) chains are indicated on the left. The form
of hsp90 included is listed at the top, along with the GA
status of the sample. Controls without added hsp90 (No
hsp90) or without immunoadsorbed PR (No PR) are also
shown. B, the corresponding levels of bound
[3H]progesterone from the samples in A are
shown (exact counts are shown at the top of each bar).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S or ATP plus molybdate
over ATP alone. Although hsp90 association with the co-chaperones p23
and Hop stabilizes its ATP-bound and nucleotide-free conformations, respectively (see above and Ref. 9), it is not clear if these associations alter hydrolysis activity or the binding and exchange of
nucleotides. In a recent study using hsp90 mutants comparable to ours,
Obermann et al. (37) also showed that the hsp90/p23 interaction required ATP binding, but not hydrolysis. In addition, they
found that p23 did not affect the ATPase activity of hsp90 under their
conditions. On the other hand, Prodromou et al. (64) have
recently shown that the ATPase activity of hsp90 is inhibited by the
yeast Hop homolog, Sti1. It will be important to study the functional
consequences of these protein-protein interactions in more detail.
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ACKNOWLEDGEMENTS |
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We acknowledge Sherry Linander for assistance in manuscript preparation and the excellent technical assistance provided by Nancy McMahon, Bridget Stensgard, and William Sullivan. SF9 cell growth, treatment, and harvesting were conducted by Dean Edwards and Kurt Christenson at the University of Colorado Cancer Center Tissue Core. We thank Timothy Haystead for generously providing ATP-Sepharose for these studies.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK46249 and by Specialized Cooperative Center in Reproductive Research Grant U54 HD 09140.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, Mayo Clinic, 200 First St. S.W., Rochester, MN
55905. Tel.: 507-284-8401; Fax: 507-284-2053; E-mail: toft.david{at}mayo.edu.
2 J. P. Grenert and D. O. Toft, unpublished results.
3 B. D. Johnson and D. O. Toft, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
hsp, heat
shock protein;
PR, progesterone receptor;
GA, geldanamycin;
Hop, hsp
organizing protein;
ATPS, adenosine
5'-O-(thiotriphosphate);
IB, incubation buffer;
PAGE, polyacrylamide gel electrophoresis;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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