(Received for publication, December 26, 1995; and in revised form, February 7, 1996)
From the
Hsp90, one of the most prominent proteins in eucaryotic cells under physiological and stress conditions, chaperones protein folding reactions in an ATP-independent way. Surprisingly, ATP binding and ATPase activity of Hsp90 has been reported by several groups. To clarify this important issue, we have reinvestigated the potential ATP binding properties and ATPase activity of highly purified Hsp90 using a number of different techniques.
Hsp90 was compared to the well characterized ATP-binding chaperone Hsc70 and to two control proteins, immunoglobulin G and bovine serum albumin, that are known to not bind ATP. Hsp90 behaved very similarly to the non-ATP-binding proteins and very differently from the ATP-binding protein Hsc70. Like bovine serum albumin and immunoglobulin G, Hsp90 (i) did not bind to immobilized ATP, (ii) could not be specifically photocross-linked with azido-ATP, (iii) failed to exhibit significant changes in intrinsic protein fluorescence upon ATP addition, and (iv) did not bind to three fluorescent ADP analogues. In contrast, Hsc70 strongly bound ATP and ADP, specifically cross-linked with azido-ATP, and exhibited major shifts in fluorescence upon addition of ATP. Finally, reexamination of the amino acid sequence of Hsp90 failed to reveal any significant homologies to known ATP-binding motifs. Taken together, we conclude that highly purified Hsp90 does not bind ATP. Weak ATPase activities associated with Hsp90 preparations may be due to minor impurities or kinases copurifying with Hsp90.
Cells respond to external stresses such as a sudden increase in
temperature with the synthesis of a distinct set of proteins called
heat shock or stress proteins (Nover, 1991). The predominant classes of
stress proteins including GroE, Hsc70, ()Hsp90, and small
Hsps have been implicated in protein folding as molecular chaperones
(Morimoto et al., 1994; Jakob and Buchner, 1994; Buchner,
1996). While the precise molecular mechanism of these chaperones is
still under extensive investigation, the ATP dependence of
chaperone-mediated protein folding is clearly the hallmark of the GroE
and Hsc70 class of stress proteins. Both the ATPase activity of these
proteins and its influence on assisted protein folding have been
analyzed in detail (cf. Morimoto et al., 1994). In contrast,
conflicting evidence exists concerning Hsp90's ATP binding
properties and ATPase activity. Hsp90 is one of the most abundant
proteins in the eucaryotic cell, even at physiological conditions. In
complex with other effector proteins such as Hsc70 and prolyl
isomerases, Hsp90 has been implicated as a molecular chaperone in the
maturation of specific protein substrates such as steroid receptors and
kinases in vivo (Pratt, 1993; Smith et al., 1993;
Jakob and Buchner, 1994; Buchner, 1996). Interestingly, assembly of
these complexes with substrate proteins has been found to be
ATP-dependent (Pratt, 1993; Smith et al., 1993). However,
since Hsc70 is also involved in the formation of these high molecular
weight assemblies, it is not yet clear which of the Hsps is responsible
for the ATP requirement. More recently, using in vitro folding
and unfolding assays, it has been demonstrated that Hsp90 may be a
general cytosolic chaperone under physiological (Wiech et al.,
1992; Shaknovich et al., 1992; Shue and Kohtz, 1994) and heat
shock conditions (Jakob et al., 1995a; Schumacher et
al., 1994). These chaperone functions as well as interactions of
Hsp90 with estrogen receptors (Inano et al., 1994) were found
to be ATP-independent. In contrast, binding of ATP to Hsp90 has been
reported (Csermely and Kahn, 1991). Binding of ATP was suggested to
result in conformational changes of Hsp90 (Csermely et al.,
1993), which in turn would affect the interaction with other proteins
(Kellermayer and Csermely, 1995). In addition evidence was presented
suggesting that some purified Hsp90 preparations exhibit potent
peptide-stimulated ATPase activity with high turnover numbers (Nadeau et al., 1992; 1993). However, Hsp90 purified from other
species did not show ATPase activity (Wiech et al., 1993;
Nardai et al., 1995).
Whether Hsp90 acts in an ATP-dependent or independent way is of crucial importance for understanding the molecular mechanism of this chaperone. Therefore we have examined the ATP binding properties of Hsp90 in detail using several independent experimental approaches. By including known ATP-binding or non-ATP-binding proteins as positive and negative controls, we were able to show that several of the methods employed previously are not suited to unambiguously demonstrate ATP binding. Those methods that turned out to be reliable demonstrate that Hsp90 does not bind ATP.
Prior to
injection, the protein was denatured to dissociate any bound
nucleotides by the addition of 2 µl of 1 M HClO to 20 µl of protein solution. The solution was then kept on
ice for 1 min, and 28 µl of 2 M potassium acetate was
added to reach neutral pH. The sample was then centrifuged at 5000 rpm
for 1 min, and 10 µl of the supernatant was analyzed for nucleotide
content as described above.
where F is the corrected fluorescence
and F
is the observed fluorescence after
subtracting the background light intensity, L
is
the total ATP concentration, and
is the absorption
coefficient of ATP (Birdsall et al., 1983).
The fluorescent nucleotides were added to 1 ml of buffer at the concentrations indicated. Subsequently, protein was added and mixed manually. All data are volume-corrected averages of 10 readings of 1-s intervals of photon counting.
The nucleotide analogues
3`-O-N-methyl-anthraniloyl diphosphate (MANT-ADP), N-8-(4-N`-methylanthranylaminobutyl)-8-aminoadenosine
diphosphate (MABA-ADP), and etheno-ADP (-ADP) were employed to
analyze binding of adenosine nucleotides to Hsp90. The concentrations
used were 0.4 µM for the nucleotides and up to 0.85
µM for Hsp90. The emission wavelengths of
-ADP,
MABA-ADP, and MANT-ADP were 410, 422, and 440 nm, respectively, and the
according excitation wavelengths were 300, 340, and 360 nm.
In
control experiments binding of MABA-ADP to the molecular chaperone DnaK
(Hsc70 from E. coli) was determined by fluorescence. In
contrast to MANT-ADP, this nucleotide analogue binds with high affinity
to DnaK. ()The conditions used were: 0.4 µM MABA-ADP, 50 mM Tris/HCl, pH 7.5, 100 mM KCl, 5
mM MgCl
, 2 mM EDTA, 2 mM dithioerythritol, 25 °C. The emission wavelength was 422 nm,
and the excitation wavelength was 340 nm.
For competition experiments, 1 mM non-labeled ADP was added to the respective protein-ADP analogue incubation reaction and the fluorescence was monitored.
Figure 1:
Influence of MgATP on
the chaperone activity of bovine Hsc70 and bovine Hsp90. CS (15
µM) was diluted 1:200 into 40 mM HEPES-KOH, pH
7.5, at 43 °C and thermal aggregation was monitored by light
scattering. Thermal aggregation of CS in the absence of additional
components (), in the presence of 0.3 µM Hsc70 with
(
) and without (
) 1 mM MgATP and in the presence
of 0.15 µM Hsp90 with and without 1 mM MgATP
(
).
Figure 2:
Binding of yeast Hsp90 and bovine Hsc70 to
ATP-agarose. 200 µg of yeast Hsp90 (A) or bovine Hsc70 (B) were mixed with 5 mg of IgG in buffer A containing 5
mM MgCl, and these were loaded on to 1 ml of
ATP-agarose preequilibrated in the same buffer. Samples were analyzed
by SDS-PAGE. A, lane 1, load; lane 2,
supernatant; lanes 3 and 4, wash 1; lanes 5 and 6, wash 2; lanes 7-9, ATP elution. B, lane 1, load; lane 2, supernatant; lanes 3 and 4, wash 1; lanes 5-7, wash
2; lanes 8-10, ATP elution. Heavy and light IgG chains
are denoted by H and L,
respectively.
As shown in Fig. 3, cross-linking of azido-ATP to Hsp90 was observed under the conditions used. However, prior to cross-linking we could not compete for azido-ATP binding by addition of a large excess of cold ATP. This shows that azido-ATP and ATP bind to different sites on Hsp90, or that azido-ATP binds with a much higher affinity than normal ATP, or that azido-ATP simply cross-links nonspecifically with Hsp90. Thus the photocross-linking of azido-ATP to Hsp90 cannot be taken as a reliable indicator of ATP binding. This view was reinforced by the finding that BSA (Fig. 3) and Fab fragment (data not shown) photocross-linked to azido-ATP in a manner similar to Hsp90. In contrast, the azido-ATP labeling observed with Hsc70 could be competed with micromolar concentrations of cold ATP.
Figure 3:
Cross-linking of Hsp90, BSA and Hsc70 with
8-azido-[-
P]ATP. 5 µg of Hsp90 (A), 5 µg of BSA (B), or 5 µg Hsc70 (C) were incubated with
8-azido-[
-
P]ATP in the presence of
increasing concentrations of non-labeled ATP. Lanes 1-8 represent cross-linking in the presence of 0, 0.05, 0.25, 0.5, 1,
3, 5, and 10 mM non-labeled ATP.
Since it is known that long-lived reaction intermediates form upon the UV-activation of azido-ATP (Todd et al., 1995), the photocross-linking of azido-ATP with Hsp90 needs to be interpreted with extreme caution.
Figure 4:
Influence of MgATP on GdmCl-induced
unfolding of yeast Hsp90. GdmCl-induced unfolding of yeast Hsp90 (50
µg/ml) was performed in the presence () or absence (
) of
2 mM ATP in buffer B. To monitor unfolding of yeast Hsp90, the
fluorescence signal at 325 nm was recorded.
We found that addition of increasing concentrations of ATP results in a decrease of the intrinsic protein fluorescence independent of the protein used. This is due to the ``inner filter effect'' of ATP, i.e. the light absorption of ATP leading to a decreased fluorescence emission. It is possible to correct fluorescence spectra recorded in the presence of ATP for this nonspecific effect of the nucleotide, as described by Birdsall et al.(1983). This allows to detect potential specific influences of ATP on the intrinsic fluorescence of proteins. After correction, as shown in Fig. 5, a slight decrease in fluorescence accounting for about 5% of the signal was observed with all proteins studied. Most importantly, however, both Hsp90 and the non-ATP-binding protein Fab gave a constant fluorescence signal at ATP concentrations, ranging from 2 µM to 2.5 mM, while the intrinsic fluorescence of Hsc70 increased by more than 10%, reflecting the specific binding of ATP. In the case of BSA, the amplitude decreased by about 25% upon increasing ATP concentrations, most likely due to unspecific interaction with ATP.
Figure 5:
Influence of ATP on the intrinsic
fluorescence of Hsp90, Hsc70, BSA, and IgG. ATP-induced fluorescence
changes of (A) 70 µg/ml bovine Hsc70 () and 57
µg/ml BSA (
), (B) 128 µg/ml yeast Hsp90
(
) and 128 µg/ml IgG (
) in the presence of various
concentrations of ATP were monitored at 335 nm (Hsc70), 330 nm (BSA),
and 325 nm (Hsp90 and IgG) after incubation for 1 min. The inner filter
effect of ATP was calculated as described under ``Materials and
Methods.''
Figure 6:
Binding of labeled ADP analogues to Hsp90
and DnaK. Titration of yeast Hsp90 against 0.4 µM MABA-ADP
() and 0.4 µM MANT-ADP (
) as well as DnaK
titration against 0.4 µM MABA-ADP (
). The
fluorescence change of the labeled analogue upon addition of the
respective protein was monitored.
, fluorescence change after
addition of 1 mM ADP in the presence of 0.75 µM DnaK and 0.4 µM MABA-ADP.
A selected set of proteins possessing either the correct type A consensus sequence or just the G-K-(T/S) tripeptide was screened for the type B consensus sequence. This motif is quite unspecific and thus is only a good indication for the presence of an ATP binding site if found together with a conserved type A sequence (Chin et al., 1988). Table 2(part B) compares the presence of type A and type B binding motifs in proteins with ATP binding properties. While proteins containing both motifs are unambiguously binding ATP, those lacking type A sequences exhibit no known ATP binding activity. Hsp90 belongs to this group of proteins, thus making it rather unlikely that Hsp90 contains an ATP binding site of the Walker type.
In recent years increasing interest has developed concerning the structure and function of the highly abundant cytosolic protein Hsp90. It is known that Hsp90 is involved in modulating the structure of target proteins, acts in concert with several heat shock and non-heat shock proteins in vivo and functions as a general molecular chaperone in vitro (cf. Jakob and Buchner, 1994). However, no consensus has yet been reached concerning the question whether or not ATP binding plays a role in the functional mechanism of Hsp90. While the in vitro chaperoning function of isolated Hsp90 is clearly ATP-independent (Wiech et al., 1992; Shaknovich et al., 1992; Shue and Kohtz, 1994; Schumacher et al., 1994; Jakob et al., 1995a; Miyata and Yahara, 1995), ATP binding and ATPase activity of Hsp90 have been reported recently (Csermely & Kahn, 1991; Csermely et al., 1993; Nadeau et al., 1992, 1993). Therefore, it is of crucial importance to study the potential influence of ATP on the structure and function of Hsp90 in more detail. We have addressed this question by studying the binding of MgATP to purified Hsp90. The first step required was to analyze whether any nucleotides were bound to purified Hsp90. Using two independent methods, isolated Hsp90 was shown to be nucleotide-free. This was a necessary prerequisite for subsequent binding studies, since the failure to detect ATP binding could therefore not be attributed to preoccupied nucleotide binding sites. Next, we reexamined experiments, the results of which had previously been interpreted as evidence for specific binding of MgATP to Hsp90. Given that Hsp90 interacts with nonnative substrate proteins as well as with native partner proteins, isolated Hsp90 can be considered to expose several reactive binding sites. Therefore, it was important to differentiate between specific binding of ATP to nucleotide binding sites and unspecific interactions of ATP with the protein surface. Based on these considerations, we compared the effects of ATP on Hsp90 with the effects of ATP on known ATP-binding and non-ATP-binding proteins. We performed cross-linking experiments of the respective proteins with azido-ATP. Specific association of MgATP could only be detected for the ATP-dependent chaperone Hsc70. The observation that cross-linking of proteins with azido-ATP can also occur in a nonspecific way is in good agreement with a previous report by Todd and co-workers(1995). Nonspecific cross-linking is based on the formation of long lived reactive azido-ATP species. In addition, we performed titration studies with labeled ADP analogues, in which the specific binding leads to a fluorescence change of the label, independent of the distribution of aromatic amino acids in the protein. With the ADP analogues used, no significant fluorescence changes could be observed upon addition of Hsp90. In contrast, DnaK, the prokaryotic Hsc70 homologue, showed large changes in fluorescence. This suggested to us that Hsp90 is not an ATP-binding protein.
Confirming these results,
we were unable to detect binding of Hsp90 to immobilized ATP and we did
not see any stabilizing effect of ATP on the unfolding transition of
Hsp90. Finally, we performed ATPase measurements with Hsp90. In
contrast to bovine Hsp90 (Wiech et al., 1993), low ATPase
activities could be detected in some yeast Hsp90 preparations. However,
ATPase assays are very sensitive to the presence of traces of highly
active, contaminating ATPases, phosphatases, and kinases, which can
result in the detection of low ATPase activities in many purified
protein preparations. One example of this is an inactive DnaK variant,
mutated so to eliminate its ATPase active site, which, even when
purified according to different protocols, gave low ATPase activities
similar to the ones obtained for Hsp90. Rates of <0.7
pmol
min
mg
apparently
represent typical background values for ATPase activities in protein
preparations. Thus extreme care is needed before low ATPase activities
can be attributed to any particular purified protein.
Based on these results and considering the analysis of the amino acid sequence, which did not reveal significant similarity with known ATP-binding motifs, we can certainly exclude the proposed tight binding of ATP to Hsp90 (cf.Table 3) as well as the high levels of ATPase activity of Hsp90, previously reported (Nadeau et al., 1992, 1993). Whether nucleotides can bind with very low affinity to Hsp90 cannot be completely ruled out based on negative results but in the light of the consistent data obtained with different assay systems this seems rather unlikely. In addition, we cannot completely exclude the possibility that binding of additional cofactors (partner proteins, peptides, transition state metals) may modulate the structure of Hsp90 to induce ATP binding. Addressing this question will require a more detailed understanding of the interaction of Hsp90 with these potential modulators.
In this context, the general question whether ATP hydrolysis is a necessary prerequisite for efficiently chaperoning protein folding arises. The increasing number of chaperones, whose function does not require ATP such as small Hsps, DnaJ, SecB, PapD, and calnexin/calreticulin argues strongly against this notion. The functional mechanism seems to vary between different members of these ATP-independent chaperones; however, all of them recognize nonnative polypeptides and are able to bind and release them in the absence of ATP. Although ATP and the co-chaperone GroES are required for the efficient release of nonnative proteins from the ATP-dependent chaperone GroEL under non-permissive folding conditions, it was recently demonstrated that the underlying cycles of binding and release are independent of ATP (Schmidt et al., 1994; Sparrer et al., 1996). Specifically, ATP decreases mainly the microscopic on-rate of GroEL for nonnative protein, which results in an efficient release of GroE-bound protein because rebinding is prevented (Sparrer et al., 1996).
In the case of Hsp90, binding cycles for
nonnative protein similar to the observed ATP-independent binding
cycles of GroEL have been postulated (Jakob et al., 1995a). It
was further suggested that Hsp90 interacts preferentially with
structured nonnative proteins. The conversion to the native form can be
induced either by ligand binding (in the case of receptors), by
myristylation (in the case of kinases) or by a folding reaction (in the
case of folding and unfolding intermediates). Since external factors
seem to drive the final conversion of the substrate protein, modulation
of the binding cycles of Hsp90 by ATP is not required. Simply binding,
release, and rebinding are sufficient to exert the Hsp90 function. In
agreement with this hypothesis, all in vitro studies with
purified Hsp90 showed that ATP is not required for the functional
interaction of Hsp90 with nonnative proteins such as citrate synthase,
MyoD, casein kinase II, and luciferase (Wiech et al., 1992;
Jakob et al., 1995a, 1995b; Shaknovich et al., 1992;
Miyata and Yahara, 1995; Schumacher et al., 1994). However,
considering the action of Hsp90 as a member of the so-called
``super chaperone complex,'' what is then the role of Hsc70
and the partner proteins? Little is known about their function, but it
was suggested that Hsc70 together with p60 mediates the formation of
the Hsp90Hsp56
p23
steroid receptor complex. The
mechanism of this ATP-driven complex formation is not yet understood,
but Hsc70 seems to be the likely candidate responsible for the observed
ATP requirement of this process. Modulators and cofactors like
molybdate may also be involved (Johnson and Toft, 1995). Additionally,
the idea has been proposed that Hsp90 and the Hsc70/DnaJ system share
functions in the folding of proteins. In this scenario, the role of the
ATP-independent Hsp90 would be to ``hold'' the nonnative
protein, while Hsc70 in cooperation with DnaJ would ``fold''
the protein in an ATP-dependent way (Bohen et al., 1995).