From the
Hsp90 is a very abundant molecular chaperone that apparently
helps to protect cellular proteins from denaturation upon temperature
upshift. The unusual ability of Hsp90 to function under conditions
where other proteins unfold prompted us to investigate the stability
and structural organization of Hsp90 itself. Both procaryotic and
eucaryotic members of the Hsp90 family were found to have very similar
physicochemical properties: (i) they are stable against thermal
unfolding up to at least 50 °C, (ii) they show biphasic, reversible
unfolding transitions in guanidinium chloride, and (iii) their
oligomerization state is strongly and rapidly affected by millimolar
concentrations of divalent cations. In the presence of MnCl
The exposure of organisms to elevated temperatures results in
the overexpression of a specific subset of polypeptides, which have
been termed heat shock proteins (Tissières et al.,
1974; Lindquist and Craig, 1988). They can be grouped in four classes,
by molecular mass and homology: the small heat shock proteins (sHsps)
with a molecular mass ranging from 15 to 32 kDa, the 70-kDa Hsps
(Hsp70, DnaK), the 60-kDa Hsps (Hsp60, GroEL), and the 90-kDa proteins
with a molecular mass ranging from 69 kDa in Escherichia coli (HtpG) to 90 kDa in humans (Hsp90) (Lindquist and Craig, 1988;
Gething and Sambrook, 1992). All four classes are known to function as
molecular chaperones and are thought to protect cells from the damaging
effects of unphysiological high temperatures (for reviews, see Gething
and Sambrook(1992), Jakob and Buchner(1994), Arrigo and Landry (1994),
Parsell and Lindquist(1994), and Hendrick and Hartl(1993)).
Members
of the Hsp90 family are the most abundant proteins in yeast under heat
shock conditions, but also constitute up to 2% of the total cellular
protein in yeast and various other cells during growth at normal
temperatures (Lai et al., 1984; Lindquist and Craig, 1988).
While eucaryotic Hsp90s have been extensively studied in relation to
their interaction with steroid receptors and kinases (Smith, 1993;
Stancato et al., 1993; Miyata and Yahara, 1992; Xu and
Lindquist, 1993), only little is known about the function of Hsp90
under heat shock conditions and only a few studies have examined E.
coli Hsp90 (Bardwell and Craig, 1987, 1988; Spence and
Georgopoulos, 1989).
The Hsp90 family is one of the most highly
conserved known; all members from bacteria to man share at least 40%
amino acid identity with each other (Bardwell and Craig, 1987;
Lindquist and Craig, 1988). The most predominant differences between E. coli and eucaryotic Hsp90s is (i) that E. coli Hsp90 is not essential, whereas yeast Hsp90 is essential; and (ii) E. coli Hsp90 lacks two charged domains (Bardwell and Craig,
1988; Borkovich et al., 1989). Given the abundance and
apparent importance of Hsp90 it is surprising how little is known about
the structural organization and stability of this chaperone, compared
with the extensive information that has been gleaned about other well
conserved chaperones such as Hsp70 and GroEL (cf. Hendrick and
Hartl(1993)). The stability of Hsp90 is of particular interest, since
heat shock proteins are thought to function under conditions where
other proteins unfold. We set out to determine and compare the
molecular organization of eucaryotic and procaryotic Hsp90 using
spectroscopic techniques and chemical cross-linking. We could
demonstrate that all members of the Hsp90 family tested have similar
physicochemical properties and that addition of divalent cations causes
major changes in the quaternary structure which are accompanied by the
instantaneous loss of function of Hsp90 as a molecular chaperone.
The UV spectra of the different Hsp90s show that the
preparations are nucleotide-free indicated by a typical A
The far-UV CD spectra of yeast and E.
coli Hsp90 showed an ellipticity maximum below 200 nm and two
minima at 208 and 220 nm (Fig. 1A) in good agreement
with data obtained for Hsp90 from higher eucaryotes (Csermely et
al., 1993). The secondary structure predicted for murine Hsp90
gives an average composition of 36%
Comparison of the GdmCl- and
thermal-induced transition curves of Hsp90 in the absence and presence
of divalent cations should provide further insight in their influence
on structure and stability of Hsp90. Although the GdmCl-induced
transition of E. coli Hsp90 in the presence of MnCl
Two general conclusions can be drawn from our studies: 1)
eucaryotic and procaryotic members of the Hsp90 family are very similar
in their physicochemical properties, although they are derived from
widely divergent organisms. These similarities mirror the high degree
of conservation in the proteins concerning sequence, structure, and
function. 2) The oligomeric state of Hsp90 is strongly affected by the
addition of millimolar quantities of divalent cations and is
accompanied by the simultaneous and rapid loss of activity. These
association forms are aggregation-sensitive and probably represent
inactive precursors of higher aggregates of Hsp90.
Yeast and E.
coli Hsp90 have similar secondary and quaternary structure as
measured by various physicochemical techniques. Size exclusion
chromatography of Hsp90 under native-like conditions confirmed earlier
experiments that suggested eucaryotic and procaryotic proteins may
elute as higher oligomeric species (Minami et al., 1991;
Spence and Georgopoulos, 1989). Since addition of small amounts of
GdmCl leads to the dissociation of Hsp90 into dimers, it seems likely
that the formation of higher oligomers of Hsp90 is due to weak
interactions between the individual dimers. This is in agreement with
earlier studies suggesting the dimer to be the native state of
association (Welch and Feramisco, 1982; Koyasu et al., 1986;
Radanyi et al., 1989; Minami et al., 1991). Since
Hsp90 is fully active under conditions under which it elutes as a
dimeric protein as well as under conditions where the elution profile
reveals an oligomeric protein (Wiech et al., 1992; Jakob et al., 1995), in contrast to other Hsps such as GroEL and
GroES, oligomerization is clearly not essential for function.
Heat
shock proteins are known to function under conditions under which other
proteins unfold. It was therefore not surprising to find that yeast and E. coli Hsp90 unfold at temperatures that are more than 15
°C above the maximal growth temperature of the organisms.
GdmCl-induced unfolding of yeast and E. coli Hsp90, which was
monitored by following changes in the tertiary structure, exerted a
biphasic transition curve with a plateau ranging from 1.4 to 1.9 M GdmCl, suggesting the formation of some unfolding intermediate. In
contrast to thermal denaturation, the GdmCl-induced unfolding of yeast
and E. coli Hsp90 seemed to be reversible processes, as
monitored by fluorescence, CD, and gel filtration.
The observation
that the cross-linking pattern of both proteins changed significantly
in the presence of divalent cations prompted us to investigate the
influence of ions on the structural organization and stability of
Hsp90. Only 20% of the proteins could be cross-linked as dimeric
proteins. The appearance of higher oligomeric species was dependent on
the concentration of divalent cations in the incubation reaction. At
the concentration used, GA is in the monomeric form and can covalently
link arginine and lysine residues which are about 8 Å apart
(Kawahara et al., 1992; Peters and Richards, 1977). The
different cross-linking pattern of Hsp90 as a result of the incubation
with divalent cations can therefore be interpreted in two ways: (i) the
divalent cation-induced conformational changes result in different
oligomeric states or (ii) the cations change the conformation of the
subunits of the oligomer so that two residues are brought in the
appropriate distance for successful cross-linking. A similar influence
of divalent ions on the cross-linking pattern of GroEL was recently
observed (Azem et al., 1994). Divalent cations affect the
structure of GroEL subunits at contact sites and therefore increase the
velocity of intra- and intermolecular (between the GroEL-7 mers)
cross-linking with GA. These divalent cation-induced changes in the
conformation of GroEL are distinct from the influence of MgCl
An in vivo relevance of divalent
cations on the function of Hsp90 is suggested by the finding that the
functional activity of Hsp90 as a molecular chaperone in vitro is dramatically reduced in the presence of divalent cations. Yeast
and E. coli Hsp90 are normally able to decelerate the thermal
inactivation of citrate synthase by binding to early unfolding
intermediates of the enzyme thus reducing the amount of
aggregation-sensitive intermediates (Jakob et al., 1995). In
the presence of divalent cations, this function of Hsp90 was almost
completely inhibited. This was true for yeast and E. coli Hsp90 as well as for Hsp90 from higher eucaryotes. Addition of
MgCl
The cation-induced release of folding
intermediates from Hsp90 suggests an in vivo regulatory role
of these ions in the function of the chaperone. It remains to be seen
if this effect of cations on the function of members of the Hsp90
family extends to other chaperones such as Hsp70 and the small Hsps.
5 µg of yeast
Hsp90 were incubated in various concentrations of GdmCl for 24 h at 20
°C and were loaded onto a Superose 6 column (25 ml) which was
equilibrated with the respective concentrations of GdmCl in 40 mM HEPES-KOH, 20 mM KCl. The flow rate was 20 ml/h.
We thank S. Lindquist for the generous gift of the
Hsp90 overexpressing yeast strain and M. Ehrmann for the polyclonal
antibodies against E. coli Hsp90. We thank H. Lilie for
critically reading the manuscript and R. Rudolph and R. Jaenicke for
continuous interest in our work.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and MgCl
defined changes in the quaternary structure
of Hsp90 could be observed which resulted in a decrease in
thermostability and an increased tendency to form larger aggregates.
The addition of divalent cations also almost completely abolished the
chaperone function of Hsp90 and induced release of folding
intermediates of citrate synthase bound to Hsp90. These modulating
effects of divalent cations on structure and function of Hsp90 in
vitro represent a potential mechanism for regulation of Hsp90
chaperone action in vivo.
Proteins
Hsp90 from bovine pancreas was purified
as described (Wiech et al., 1993). The protein concentration
was determined according to Bradford(1976), using bovine serum albumin
as a standard. Hsp90 from yeast and E. coli were purified as
described below. Mitochondrial citrate synthase (EC 4.1.3.7.) was
obtained from Boehringer Mannheim GmbH (Mannheim, Germany). For citrate
synthase the published extinction coefficient of 1.78 for a 1 mg/ml
solution in a 1-cm cuvette was used (West et al., 1990).
Citrate synthase was stored in 50 mM Tris, 2 mM EDTA,
pH 8.0. Concentrations of citrate synthase and Hsp90 are given for the
dimeric form (Minami et al., 1994). GroEL was the gift of Dr.
Marion Schmidt (University of Regensburg, Germany). The monoclonal
antibody against Hsp90 from yeast was from StressGen (Victoria, British
Columbia, Canada). The polyclonal antiserum against E. coli Hsp90 was the kind gift of Dr. Michael Ehrmann (University of
Konstanz, Germany).
Chemicals
The chemicals used for polyacrylamide
gel electrophoresis and the hydroxyapatite resin were obtained from
Bio-Rad. Diethylaminoethyl (DE52)-cellulose, S-Sepharose, and Superose
6 were from Pharmacia (Uppsala, Sweden). Guanidinium chloride
(GdmCl)(
)ultrapure and HEPES were purchased from
ICN (New York). Glutaraldehyde (GA, 25% (v/v) in H
0) and
PMSF was obtained from Serva (Heidelberg, Germany). PABA, leupeptin,
pepstatin, oxalacetic acid, acetyl-CoA, and a standard protein mix for
size exclusion chromatography were purchased from Boehringer Mannheim
GmbH. Nile red was obtained from Eastman Kodak. MnCl
and
(NH
)
Mo
O
were obtained
from Sigma.
Buffers
Buffer A: 40 mM HEPES-KOH, 5
mM EDTA, 1 mM 1,4-dithioerythritol, 5% (v/v)
glycerol, pH 7.5; buffer B: 20 mM sodium phosphate, pH 5.5;
buffer C: 100 mM potassium phosphate, 5% (v/v) glycerol, pH
6.8; buffer D: 40 mM HEPES-KOH, 400 mM KCl, pH 7.5;
buffer E: 40 mM HEPES-KOH, 20 mM KCl, 5% (v/v)
glycerol, pH 7.5; buffer 1: 50 mM potassium phosphate, 20
mM KCl, 5% (v/v) glycerol, 1 mM EDTA, pH 7.5; buffer
2: 50 mM HEPES-KOH, 20 mM KCl, 5% (v/v) glycerol, 1
mM EDTA, pH 7.5; buffer 3: 20 mM potassium phosphate,
5% (v/v) glycerol, pH 6.8; buffer 4: 10 mM MES-HCl, 20 mM KCl, 5% (v/v) glycerol, pH 5.5.
Purification of Yeast Hsp90
Yeast Hsp90 was
purified from a hsc82 and hsp82 deletion mutant
strain ECUpep4 that carried hsc82 on a 2µ URA3
overproducing plasmid (a kind gift of S. Lindquist). The yeast cells
were grown at 25 °C in YPD medium (1% (w/v) yeast extract, 2% (w/v)
Bacto-peptone, 2% (w/v) D-(+)-glucose) to an A of 2.8, harvested, and washed twice in buffer
A, containing a protease inhibitor mix (1 mM PMSF, 2.5 mM PABA, 1 µg/ml leupeptin, and 1 µg/ml pepstatin). All
following steps of the Hsp90 purification were carried out at 4 °C
and the protease inhibitor mix was added during the first two steps of
the purification. Cells were lysed with glass beads, adjusted to 0.1 M NaCl, centrifuged (48,000
g, 60 min, 4
°C), and the supernatant was loaded onto a DE52 column equilibrated
in buffer A containing 0.1 M NaCl. The column was washed with
buffer A (+ 100 mM NaCl), and Hsp90 was eluted between
200 and 300 mM NaCl in a linear salt gradient. After
centrifugation, the dialyzed (buffer B) Hsp90 pool was loaded onto
S-Sepharose, equilibrated in buffer B. The major part of Hsp90 could be
detected in the flow-through. The protein was concentrated (Amicon,
YM30 membrane) and dialyzed against buffer C. It was then further
purified on a hydroxyapatite column equilibrated in buffer C. Hsp90
eluted between 150-200 mM potassium phosphate.
Concentrated fractions adjusted to 400 mM KCl were applied
onto a Superose 6 column and eluted with buffer D. Hsp90 fractions were
concentrated, dialyzed against buffer E, and stored at -70
°C. All purification steps were monitored by SDS-PAGE and
immunoblots, using monoclonal antibodies against Hsp90. Yeast Hsp90 was
about 98% pure as determined by densitometry.
Cloning and Purification of E. coli Hsp90
E.
coli Hsp90 was expressed from its own heat shock promoter using
the plasmid pBJ935. This plasmid contains the E. coli Hsp90
gene (htpG) cloned into the high copy number plasmid pUC19. It
was constructed by cloning a 2.5-kilobase pair SalI-EcoRI fragment containing the Hsp90 gene into
the PstI-EcoRI sites of pUC19. This fragment contains
DNA extending from the SalI site 165 base pairs upstream from
the E. coli Hsp90 initiation codon to the EcoRI site
0.5 kilobase downstream from the termination codon. The SalI
site was first ligated to a PstI site by cloning a kanamycin
resistance cassette from pUC-4K into the SalI site upstream
from E. coli Hsp90 as described previously (Bardwell and
Craig, 1987). This construct was introduced into the wild type E.
coli strain MG1655 to generate the strain JCB 867. This strain
grew normally at 30 °C and overproduced E. coli Hsp90 to
>30% of the cellular protein. Expression at 37 °C or higher
temperatures resulted in rapid plasmid loss. JCB 867 cells were grown
in LB medium in the presence of 100 µg/ml ampicillin for 20 h at 30
°C to an A of 4.7. The cells were harvested
by centrifugation and resuspended in buffer 1, containing 2 mM PMSF. All further purification steps were performed at 4 °C. E. coli Hsp90 was identified on SDS-PAGE and immunoblots with
polyclonal antibodies against E. coli Hsp90 (provided by M.
Ehrmann). The following purification protocol is based on the protocol
originally used by Spence and Georgopoulos(1989). The cells were lysed
by using French press at 18,000 p.s.i. for three cycles. The crude
extract was centrifuged (48,000
g, 30 min, 4 °C).
Ammonium sulfate was added to a final concentration of 60% (w/v), and
the lysate was incubated on ice for 30 min. The pellet of the ammonium
sulfate precipitation was resuspended in buffer 2, containing 1 mM PMSF. After dialysis against buffer 2 and centrifugation, the
supernatant was loaded onto a DE52-cellulose column, equilibrated in
buffer 2. The proteins were eluted with a KCl gradient. E. coli Hsp90 eluted from the column between 150 and 300 mM KCl.
The concentrated and dialyzed (buffer 3) pool was loaded onto a
hydroxyapatite column equilibrated in buffer 3. E. coli Hsp90
eluted between 50 and 150 mM in a 20-200 mM potassium phosphate gradient. The pH of the protein solution was
adjusted to 5.5 by dialysis against buffer 4. After centrifugation the
supernatant was loaded onto a DE52 column, equilibrated in buffer 4. E. coli Hsp90 eluted at 150-250 mM KCl in
buffer 4. Purified E. coli Hsp90 was nearly homogeneous as
judged by SDS-PAGE (>98%). Finally, 80% (w/v) ammonium sulfate was
added to the protein solution, and the ammonium sulfate precipitate was
stored at 4 °C.
Determination of Protein Concentration
The protein
concentration of yeast and E. coli Hsp90 was determined using
the extinction coefficient of 0.72 and 1.25, respectively, given for a
1 mg/ml solution in a 1-cm cuvette. The extinction coefficients of the
proteins were calculated according to Wetlaufer(1962) by using the
aromatic amino acid composition of the proteins predicted from the DNA
sequence. The determined protein concentrations could be confirmed
using the tryptophan titration method for denatured proteins (Payot,
1975).
ratio of 1.75 for yeast Hsp90 and 1.83 for E. coli Hsp90.
Circular Dichroism Measurements
Near- and far-UV
circular dichroism (CD) spectra were recorded in a Jasco J 500 A
spectropolarimeter equipped with a Jasco DP 500 N data processor. Yeast
and E. coli Hsp90 were dialyzed overnight against 40 mM HEPES-KOH, pH 7.5. Near-UV CD spectra were recorded from 250 to
350 nm in thermostatted 1-cm quartz cuvettes at 8 °C. The far-UV CD
spectra were recorded from 200 to 250 nm at room temperature in 0.01-cm
quartz cuvettes. All spectra were base line-corrected. To determine the
influence of GdmCl on the secondary structure of Hsp90, the CD signal
was recorded at 220 nm in 0.01-cm quartz cuvettes. The influence of
ions on the far-UV CD spectrum of E. coli Hsp90 was monitored
after incubating the samples in the presence of the respective ions for
30 min at 20 °C. Mean residue ellipticities for near- and far-UV CD
spectra were calculated based on a mean residue molecular weight of
112.
Fluorescence and Light Scattering Measurements
The
unfolding and refolding transitions of yeast and E. coli Hsp90
were monitored by measuring the change in intrinsic fluorescence. The
measurements were performed in a Perkin-Elmer MPF44A luminescence
spectrometer at 20 °C. The individual spectra were recorded from
290 to 350 nm in 1-cm quartz cells at an excitation wavelength of 285
nm. The spectral bandwidths were 5 and 10 nm for excitation and
emission, respectively. To monitor the interaction of the dye nile red
with accessible hydrophobic surfaces of the protein, fluorescence
spectra were recorded from 560 to 700 nm at an excitation wavelength of
550 nm. The spectral bandwidths were 5 and 15 nm for excitation and
emission, respectively. E. coli Hsp90 (0.1 mg/ml in 40 mM HEPES-KOH, pH 7.5) was incubated in the presence of 5 mM KCl, MgCl, MnCl
,
(NH
)
Mo
O
or in the
absence of ions for 30 min at room temperature. Nile red (0.25 mM in Me
SO) was diluted 1:250 into the protein solutions
and the samples were incubated for 5 min at room temperature before
recording the spectra. To monitor the influence of MgCl
on
the stability of bovine and yeast Hsp90, light scattering measurements
were performed. Both excitation and emission wavelengths were set to
360 nm with 3 nm slit widths. Bovine and yeast Hsp90 (25 µg/ml)
were incubated in various concentrations of MgCl
in 40
mM HEPES-KOH, pH 7.5, for 24 h at 20 °C.
GdmCl-induced Unfolding and Refolding of Yeast and E.
coli Hsp90
GdmCl-induced unfolding of yeast and E. coli Hsp90 was performed by dilution of the proteins into various
concentrations of GdmCl (40 mM HEPES-KOH, pH 7.5) ranging from
0 to 6 M. The respective protein concentrations are given in
the figure legends. The samples were incubated for 24 h at 20 °C to
achieve equilibrium. To study the influence of divalent ions on the
stability of E. coli Hsp90, GdmCl-induced unfolding was
performed as described in the presence and absence of 5 mM MnCl. For refolding experiments, yeast and E. coli Hsp90 was first denatured in 6 M GdmCl (40 mM HEPES-KOH, pH 7.5) for 6 h at 20 °C. Then the unfolded
proteins were refolded by diluting into various concentrations of the
denaturant ranging from 0 to 6 M GdmCl and further incubated
for 24 h at 20 °C.
Temperature-induced Unfolding and Aggregation of Yeast
and E. coli Hsp90
To monitor thermal unfolding and aggregation
of yeast and E. coli Hsp90, fluorescence and light scattering
measurements were performed in the temperature range from 30 °C to
75 °C. The measurements were carried out in stirred 1-cm quartz
cells in a Perkin-Elmer MPF44A luminescence spectrometer equipped with
thermostatted cell holder connected to a thermoprogrammer. Yeast and E. coli Hsp90 were diluted into 40 mM HEPES-KOH, pH
7.5, at 25 °C (final concentration: 60 µg/ml). The tryptophan
fluorescence of Hsp90 was measured at an excitation wavelength of 295
nm and an emission wavelength of 330 nm. The slit widths were set to 5
and 10 nm for emission and excitation, respectively. Light scattering
measurements were performed in a stirred cuvette with both excitation
and emission wavelength set to 360 nm, with 3 nm slit widths.
Gel Filtration of Yeast and E. coli Hsp90
For size
exclusion chromatography, an analytical Superose 6 fast protein liquid
chromatography column (25 ml) was used at a flow rate of 20 ml/h. The
eluting protein was detected by fluorescence at 330 nm with excitation
at 285 nm. The fluorescence detector (F 1000, Merck-Hitachi) was
connected to an SP 4270 Integrator (Spectra-Physics) for evaluation of
the peak areas. To determine the apparent molecular mass of yeast Hsp90
after 24 h incubation in the presence of various amounts of GdmCl, the
elution buffer (40 mM HEPES-KOH, 20 mM KCl, pH 7.5)
was supplemented with the respective GdmCl concentrations. Elution
profiles of the standard protein mix (-galactosidase, IgG, Fab,
myoglobin) were also determined at the various concentrations of
denaturants. With up to 1.5 M GdmCl present in the elution
buffer, no significant change in the mobility of the standard proteins
could be observed. To analyze the influence of MnCl
on the
mobility of yeast and E. coli Hsp90, 5 mM MnCl
was added to the sample prior to loading it onto the gel
filtration column. The elution buffer also contained 5 mM MnCl
.
Cross-linking of Yeast and E. coli Hsp90
Chemical
cross-linking of bovine, yeast, and E. coli Hsp90 was
performed using GA as the cross-linking reagent. The optimal GA
concentration was determined by titration and shown to be 20
mM. 4 µg of bovine Hsp90, yeast Hsp90, or E. coli Hsp90 were incubated in 20 µl of 40 mM HEPES-KOH, pH
7.5, for 30 min at room temperature in the presence or absence of
various ions. The concentrations of these ions are given in the figure
legends. For cross-linking, 1.6 µl 0.25 M GA (in 40 mM HEPES-KOH, pH 7.5) was added and the samples were incubated for 2
min at 37 °C. To stop the cross-linking process, the samples were
supplemented with 5 µl of 1 M Tris-HCl, pH 8.0, and
incubation was continued on ice. The cross-linked bands of Hsp90 were
visualized by 3-20% gradient SDS-PAGE and quantified by
densitometry.
Activity Assay for the Molecular Chaperone Function of
Yeast and E. coli Hsp90s
To monitor the activity of bovine,
yeast, and E. coli Hsp90 as molecular chaperones, their
influence on the thermal unfolding and aggregation process of citrate
synthase at 43 °C was monitored as described previously (Jakob et al., 1995). The effects of ions on the activity of Hsp90
were studied by diluting the ions into the Hsp90 solution prior to the
addition of citrate synthase.
Spectroscopic Properties of Yeast and E. coli
Hsp90
To gain insight into the native structure of procaryotic
and eucaryotic members of the Hsp90 family, fluorescence and circular
dichroism spectra in the far- and near-UV region were recorded. The
fluorescence spectra of yeast and E. coli Hsp90 corresponded
well to the known aromatic amino acid composition of the respective
proteins (data not shown).
-helix and 46%
-strands
(Csermely et al., 1993). Hsp90 from yeast and E. coli apparently have a similar secondary structure composition except
the
-helical content of E. coli Hsp90 may be slightly
higher.
Figure 1:
Secondary and tertiary
structure of eucaryotic and procaryotic Hsp90. A, far-UV CD
spectra of 1 mg/ml yeast Hsp90 (-) and 1 mg/ml E. coli Hsp90 (
) in 40 mM HEPES-KOH,
pH 7.5, at room temperature. B, near-UV CD spectra of 0.6
mg/ml yeast Hsp90 (-) and 0.8 mg/ml E. coli Hsp90
(
) in 40 mM HEPES-KOH, pH 7.5, at 8
°C.
Near-UV CD spectra of proteins represent a highly sensitive
criterion for the native state of a protein and as such can be used as
a ``fingerprint'' of the correctly folded conformation and
tertiary structure (Schmid, 1989). Accordingly, significant differences
in the near-UV spectra of yeast and E. coli Hsp90 could be
detected (Fig. 1B). This spectroscopic characterization
allowed us to investigate the stability of Hsp90 against chemical and
thermal unfolding.
GdmCl-induced Folding Transitions of Yeast and E. coli
Hsp90
Yeast and E. coli Hsp90 exhibited a similar
biphasic unfolding behavior in the presence of increasing
concentrations of the denaturant GdmCl as monitored by fluorescence (Fig. 2). The midpoint of the first transition occurred at about
1.2 M GdmCl, followed by a plateau ranging from 1.4 to 1.9 M GdmCl. The second transition with the midpoint at 2.2 M GdmCl resulted in a protein lacking detectable tertiary structure.
To analyze whether the loss in tertiary structure is accompanied by or
precedes the loss of secondary structure, we monitored GdmCl-induced
changes in the CD signal at 220 nm (Fig. 2A), where the
CD signal is dominated by secondary structural elements. Comparison of
the changes observed by fluorescence and CD spectroscopy showed that in
the first transition, some secondary and tertiary interactions are lost
simultaneously (Fig. 2A). For the second transition,
however, noncoincident transitions were obtained for the loss of
secondary and tertiary structure. At 4.0 M GdmCl both yeast
and E. coli Hsp90 were completely unfolded. To characterize
the unfolding of yeast Hsp90 further, gel filtration chromatography was
performed at different concentrations of GdmCl (). Under
native conditions, yeast Hsp90 eluted from the gel filtration column
with a molecular mass of about 360 kDa, which corresponds to an
apparent tetramer. High molecular weight complexes of Hsp90 eluting
from size exclusion chromatography have been demonstrated previously
for murine Hsp90 (Minami et al., 1991) and for E. coli Hsp90 (Spence and Georgopoulos, 1989). However, the prevailing
body of evidence suggests that Hsp90 is a dimer (Welch and Feramisco,
1982; Koyasu et al., 1986; Radanyi et al., 1989;
Spence and Georgopoulos, 1989; Minami et al., 1991). The
addition of 10 mM GdmCl to sample and gel filtration buffer
resulted in a mobility shift from the high molecular weight oligomers
to the molecular mass of a dimeric protein (). The
formation of higher oligomeric species could be due to weak
interactions between the subunits and does not influence the functional
state of Hsp90. Dimeric yeast Hsp90 eluted from the gel filtration
column at GdmCl concentrations ranging from 0.01 to 0.8 M. At
1.5 M GdmCl the mobility of yeast Hsp90 changed, presumably
due to the partial unfolding and aggregation of the protein. To monitor
the reversibility of Hsp90 unfolding, denatured Hsp90 was diluted into
GdmCl concentrations ranging from 0 to 6 M. Besides a slight
hysteresis between the unfolding and refolding curves of yeast Hsp90,
the transitions obtained were very similar, showing that the unfolding
of yeast and E. coli Hsp90 is reversible (Fig. 2B and data not shown). Slight aggregation occurring during the
refolding of both Hsp90s decreased the yield of renatured protein.
After removal of the aggregates far-UV CD spectra and gel filtration
analysis of the refolded yeast Hsp90 revealed a fully renatured,
native-like protein (data not shown).
Figure 2:
GdmCl-induced unfolding and refolding of
eucaryotic and procaryotic Hsp90. A, structural changes during
the GdmCl-induced unfolding of E. coli Hsp90. E. coli Hsp90 (60 µg/ml in 40 mM HEPES-KOH, pH 7.5) was
incubated in various concentrations of GdmCl for 24 h at 20 °C. The
fluorescence signal at 325 nm was recorded (). For monitoring
changes in the secondary structure 1.4 mg/ml E. coli Hsp90 was
incubated in various concentrations of GdmCl for 24 h at 20 °C
before the CD signal at 220 nm was recorded (
). B,
GdmCl-induced unfolding and refolding of yeast Hsp90. For unfolding
experiments yeast Hsp90 (50 µg/ml in 40 mM HEPES-KOH, pH
7.5) was incubated in various concentrations of GdmCl for 24 h at 20
°C. To initiate refolding, denatured yeast Hsp90 (1 mg/ml in
denatured in 6 M GdmCl) was diluted 1:20 into various
concentrations of GdmCl and incubated for 24 h at 20 °C to achieve
equilibrium. To monitor unfolding (
) and refolding (
) of
yeast Hsp90, the fluorescence signal at 325 nm was
recorded.
Thermal Unfolding of Yeast and E. coli Hsp90
Since
eucaryotic and procaryotic Hsp90s are able to protect proteins from
thermal inactivation and aggregation (Jakob et al., 1995), it
was interesting to investigate the thermostability of these heat shock
proteins. We monitored tryptophan fluorescence as well as the light
scattering signal of yeast and E. coli Hsp90 in the
temperature range from 30 to 75 °C (Fig. 3). Up to 50 °C,
the tryptophan fluorescence signal of yeast Hsp90 decreased linearly
with increasing temperature (Fig. 3A), corresponding to
the decrease in specific fluorescence of tryptophan with increasing
temperature (Schmid, 1989). Between 59 and 64 °C a sharp increase
in the intrinsic tryptophan fluorescence could be observed with the
midpoint of the transition at 61 °C. This is likely to reflect the
thermal unfolding of the protein. Whether unfolding results in an
increase or decrease of fluorescence is most likely determined by
changes in the micro-environment of aromatic residues and can thus not
be interpreted further. Due to the increasing influence of aggregation,
the subsequent decrease in fluorescence at higher temperatures occurred
at a rate greater than the linear decline of free tryptophan
fluorescence at increasing temperature. Qualitatively similar results
were obtained with E. coli Hsp90 (Fig. 3). The thermal
unfolding transition of the procaryotic member of the Hsp90 family
occurred at about 64 °C (Fig. 3A) and was also
accompanied by aggregation (Fig. 3B). Hsp90 from E.
coli and yeast are thus more thermostable than Hsp90 from higher
eucaryotes where the thermal transition of Hsp90 occurred at 50 °C
(Lanks et al., 1992).
Figure 3:
Thermal unfolding and aggregation of
procaryotic and eucaryotic Hsp90. Yeast Hsp90 and E. coli Hsp90 were diluted into incubation buffer (40 mM HEPES-KOH, pH 7.5) at 25 °C. The final protein concentration
was 60 µg/ml. The heating rate was 1 °C/min. A,
thermal unfolding transition of yeast (-) and E. coli Hsp90 (
). The fluorescence signal at
330 nm was monitored. B, thermal aggregation of yeast (
)
and E. coli Hsp90 (
). To monitor aggregation the change
in light scattering signal was followed.
Influence of Divalent Cations on the Cross-linking
Pattern of Hsp90
To analyze the quaternary structure of Hsp90
under native conditions, we performed cross-linking studies with GA. In
the presence of 2 mM EDTA, only 20% Hsp90 could be
cross-linked as dimers at the given Hsp90 and optimized GA
concentration (Fig. 4). No higher cross-linked species could be
detected on the gradient gel. This is in contrast to the results,
obtained by size exclusion chromatography under native-like conditions,
but in agreement with the analysis of eucaryotic Hsp90 where mainly
dimeric species were detected (Welch and Feramisco, 1982; Koyasu et
al., 1986; Radanyi et al., 1989; Minami et al.,
1991). This confirms the hypothesis that the interactions between the
subunits responsible for the appearance of high molecular weight
complexes in gel filtration experiments are rather weak. Because it has
been speculated that Hsp90 may bind ATP (Csermely and Kahn, 1991),
comparative cross-linking experiments were performed with
nucleotide-free Hsp90 (see ``Materials and Methods'') in the
presence of 5 mM MgATP, 5 mM ATP, and 5 mM
MgCl (data not shown). While ATP and MgATP did not show any
influence on the cross-linking pattern, MgCl
shifted the
equilibrium between monomeric and dimeric Hsp90 significantly toward
higher cross-linked species, including tetramers (Fig. 4, A and C). Therefore we examined the effect of various
monovalent and divalent ions as well as transition state metals on the
association state of bovine, yeast, and E. coli Hsp90. Fig. 4A shows the cross-linking pattern of yeast Hsp90
in the presence of various amounts of KCl, MnCl
,
MgCl
, and
(NH
)
Mo
O
. Although
monovalent ions and the transition state metal molybdate had no effect
on the cross-linking pattern of Hsp90, the presence of divalent cations
increased the amount of Hsp90 cross-linked into dimers and tetramers.
MgCl
(Fig. 4, A and C) and
MnCl
(Fig. 4, A and B) had an
equivalent effect. As shown in Fig. 4B, the amount of
monomeric species decreased with raising the MnCl
concentration, whereas the amount of cross-linked dimers and
tetramers increased. Higher MnCl
concentrations resulted in
an increase in the amount of tetrameric cross-linked Hsp90 at the
expense of dimers. The change in quaternary structure could also be
confirmed by size exclusion chromatography of E. coli Hsp90.
Under native-like conditions (40 mM HEPES-KOH, 20 mM KCl) the protein eluted from a Superose 6 column as a 540-kDa
oligomer, corresponding to an apparent octamer. No monomeric or dimeric
forms were detected. The presence of 5 mM MnCl
in
the sample and the elution buffer induced a significant change in the
elution profile of E. coli Hsp90. The mobility corresponded
now to an apparent tetrameric protein (approximately 240 kDa). The
effects of divalent cations on the quaternary structure of Hsp90 from
higher eucaryotes were similar to their effects on E. coli Hsp90 (Fig. 4C). Thus, we conclude that divalent
cation-induced conformational changes represent a general property of
members of the Hsp90 family. Interestingly, the change in Hsp90
conformation seemed to be a rapid process occurring within 1 min after
addition of divalent cations (data not shown).
Figure 4:
Cross-linking of eucaryotic and
procaryotic Hsp90 with GA. A, cross-linking of yeast Hsp90 in
the presence of various concentrations of mono- and divalent cations
and the transition state metal molybdate. Yeast Hsp90 was cross-linked
with GA after 30-min incubation in the presence of 2 mM EDTA (lane 4) and increasing concentrations (1, 2, 5, and 10
mM) KCl (lanes 5-8), MnCl (lanes 9-12), MgCl
(lanes
13-16), and
(NH
)
Mo
O
(lanes
17-20). Similar results were obtained with bovine and E.
coli Hsp90. Lanes 1, 2, and 3 represent low and
high molecular weight standards and noncross-linked yeast Hsp90,
respectively. M, D, and T represent monomeric,
dimeric, and tetrameric cross-linked species. B,
MnCl
-induced oligomerization of E. coli Hsp90. E. coli Hsp90 was incubated at room temperature in the
presence of increasing amounts of MnCl
before cross-linking
with GA was performed. The formation of monomeric (
), dimeric
(
), and tetrameric (
) cross-linked species was analyzed by
densitometry of the respective bands after SDS-PAGE. C,
cross-linking pattern of bovine Hsp90 in the presence of
MgCl
. Bovine Hsp90 was incubated at room temperature in the
presence of various amounts of MgCl
before cross-linking
with GA was performed. The incubation reaction contained 2 mM EDTA (lane 1) or increasing concentrations of
MgCl
. Lanes 2 to 13 refer to 0.1, 0.25,
0.5, 0.75, 1.0, 2.5, 5.0, and 10.0 mM MgCl
in the
incubation reaction. M, D, and T represent monomeric,
dimeric, and tetrameric cross-linked
species.
Effects of Divalent Cations on Structure and Stability of
Yeast and E. coli Hsp90
To examine, whether incubation in the
presence of divalent cations induces conformational changes in the
secondary and tertiary structure of Hsp90, fluorescence, and CD
measurements were performed. No changes in the secondary structure and
tertiary structure of yeast and E. coli Hsp90 could be
detected after a 30-min incubation in the presence of 5 mM
MgCl and MnCl
(data not shown). Additional
fluorescence measurements with the hydrophobic dye nile red did not
reveal major changes in the accessible hydrophobic surfaces of the
proteins (data not shown).
was comparable with the transition in the absence of additional
components (Fig. 2A), significant differences in the
thermal stability of E. coli Hsp90 in the presence of
MnCl
could be detected (Fig. 5A). The
midpoint temperature of the thermal unfolding transition of Hsp90 in
the presence of divalent cations was far below the temperature, where
unfolding took place in the absence of ions. The cation-induced
tendency of Hsp90 to form larger aggregates could be confirmed by
measuring the light scattering of Hsp90, which was incubated for 24 h
in the presence of various amounts of MgCl
, ranging from 0
to 15 mM (Fig. 5B). This aggregation process
did not result in a major change in fluorescence, explaining the
similar fluorescence spectra in the absence and presence of divalent
cations (data not shown).
Figure 5:
Influence of divalent cations on the
stability of Hsp90.A, thermal unfolding of E. coli Hsp90 in the absence and presence of divalent cations. The changes
in tertiary structure of E. coli Hsp90 (60 µg/ml in 40
mM HEPES-KOH, pH 7.5) in the absence (
) or presence (-) of 5 mM MnCl
were
monitored by fluorescence at 330 nm. The heating rate was 0.4 °C. B, MgCl
induced aggregation of yeast and bovine
Hsp90. Light scattering measurements were performed after incubation of
25 µg/ml yeast Hsp90 (
) and 25 µg/ml bovine Hsp90 (
)
in the presence of various concentrations of MgCl
for 24 h
at 20 °C.
Influence of Divalent Cations on the Function of Hsp90 as
Molecular Chaperones in Vitro
We examined the influence of Hsp90
on the thermal aggregation and inactivation of citrate synthase at 43
°C. In the absence of divalent cations, E. coli Hsp90 was
able to suppress the thermal aggregation of citrate synthase
significantly (Fig. 6A) and to stabilize the protein by
interacting with early unfolding intermediates (Jakob et al.,
1995). However, in the presence of 1 mM MgCl the
ability of E. coli Hsp90 to function as a molecular chaperone
was almost completely abolished (Fig. 6A). Maximum
aggregation was reached within 20 min both in the complete absence of
Hsp90 or in the presence of Hsp90 and cations. Only a slight delay in
the increase of the light scattering signal of citrate synthase in the
presence of E. coli Hsp90 and cations could be observed
initially. The aggregation process of citrate synthase itself was not
affected by the presence of MgCl
(Fig. 6A).
Experiments with bovine Hsp90 gave similar results (data not shown).
Figure 6:
Divalent cation-induced change in the
chaperone activity of Hsp90 in vitro.A, influence of
MgCl on the E. coli Hsp90-induced suppression of
thermal aggregation of citrate synthase at 43 °C. 0.075 µM citrate synthase was incubated in the presence of 0.6 µME. coli Hsp90 with (
) or without (
) 1 mM MgCl
in the incubation reaction. Similar results were
obtained with yeast and bovine Hsp90. Closed squares represent
the light scattering signal of citrate synthase ± 1 mM MgCl
in the absence of 0.6 µME. coli Hsp90. B, influence of MnCl
on the yeast
Hsp90-induced stabilization of thermally inactivating citrate synthase
at 43 °C. Citrate synthase (0.075 µM) was incubated
with 0.6 µM yeast Hsp90 in the absence of divalent cations
(
) or in the presence of (
) 1 mM MnCl
of (
) 3 mM MnCl
. Control experiments
of citrate synthase without Hsp90 were also performed in the presence
of 3 mM MnCl
(
) or in the absence of
MnCl
(
). Qualitatively similar data were obtained
with E. coli and bovine Hsp90. C,
MgCl
-induced release of citrate synthase intermediates from
preformed Hsp90
citrate synthase complexes. Citrate synthase
(0.075 µM) was incubated in the absence (
) or
presence of 0.15 µM bovine Hsp90 at 43 °C. After 5 min
of incubation in the presence of Hsp90, MgCl
(5
mM) was added followed 5 s later by the addition of (
)
0.3 µM bovine serum albumin or (
) 0.1 µM GroEL (14-mer). The aggregation kinetics were monitored by light
scattering.
Confirming the results we obtained with light scattering
measurements, we also observed that Hsp90 had much less effect on the
stabilization of citrate synthase against thermal inactivation in the
presence of divalent cations than in their absence (Fig. 6B). Again, the presence of MgCl or
MnCl
in the incubation reaction had no influence on the
inactivation process of citrate synthase in the absence of Hsp90 or in
the presence of bovine serum albumin (data not shown). To examine
whether divalent cations exert also an influence on preformed
Hsp90
citrate synthase complexes, MgCl
was added 5 min
after start of the inactivation process (Fig. 6C). This
resulted in a sudden increase in the light scattering signal,
indicating that citrate synthase was immediately released from Hsp90
and aggregated. Hsp90 itself did not reveal any detectable aggregation
in the presence of divalent cations in the time range of the experiment
(data not shown). To test whether GroEL is able to recognize released
citrate synthase molecules in the presence of divalent cations, 5 s
after the addition of MgCl
GroEL was added. As shown in Fig. 6C, substoichiometric amounts of GroEL were capable
of binding these intermediates and forming a stable complex, thus
suppressing aggregation. Citrate synthase was still a substrate protein
for GroEL in the presence of divalent cations and made it thus less
likely that divalent cations induce conformational changes in citrate
synthase itself, preventing its interaction with chaperones.
on the ATPase activity of GroEL (Azem et al., 1994) and
do not affect its function. In contrast to the stabilizing effects of
divalent cations on urea-induced unfolding of GroEL, no significant
stabilization by cations of yeast and E. coli Hsp90 against
chemical denaturation was observed. Instead, incubation of Hsp90 for 24
h in the presence of various concentrations of divalent cations led to
an increasing tendency of aggregation. Furthermore, the thermal
unfolding transition of Hsp90 occurred at lower temperatures in the
presence of MnCl
. These results and gel filtration
experiments in the presence of divalent cations led to the conclusion
that divalent cation-induced changes in the conformation of Hsp90
produce different oligomeric states, which could represent precursors
of larger aggregates.
to a complex formed between Hsp90 and citrate synthase
resulted in a sudden increase in the light scattering signal. This
indicated that the cation-induced conformational changes in Hsp90 occur
very quickly and induce release of unfolding intermediates of citrate
synthase from Hsp90.
Table: Size exclusion chromatography of yeast Hsp90 in
the presence of various concentrations of GdmCl
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.