(Received for publication, March 24, 1995)
From the
We have studied the effects of the Sulfolobus solfataricus chaperonin on the aggregation and inactivation upon heating of
four model enzymes: chicken egg white lysozyme (one 14.4-kDa chain),
yeast -glucosidase (one 68.5-kDa chain), chicken liver malic
enzyme (four 65-kDa subunits), and yeast alcohol dehydrogenase (four
37.5-kDa subunits). When the proteins were heated in the presence of an
equimolar amount of chaperonin, 1) the aggregation was prevented in all
solutions; 2) the inactivation profiles of the single-chain enzymes
were comparable with those detected in the absence of the chaperonin,
and enzyme activities were regained in the solutions heated in the
presence of the chaperonin upon ATP hydrolysis (78 and 55% activity
regains for lysozyme and
-glucosidase, respectively); 3) the
inactivation of the tetrameric enzymes was completely prevented,
whereas the activities decreased in the absence of the chaperonin. We
demonstrate by gel filtration chromatography that the chaperonin
interacted with the structures occurring during thermal denaturation of
the model proteins and that the interaction with the single-chain
proteins (but not that with the tetrameric proteins) was reversed upon
ATP hydrolysis. The chaperonin had nonequivalent surfaces for the
binding of the model proteins upon heating: the thermal denaturation
intermediates of the single-chain proteins share Surfaces I, while the
thermal denaturation intermediates of the tetrameric proteins share
Surfaces II. ATP binding to the chaperonin induced a conformation that
lacked Surfaces I and carried Surfaces II. These data support the
concept that chaperonins protect native proteins against thermal
aggregation by two mechanistically distinct strategies (an
ATP-dependent strategy and an ATP-independent strategy), and provide
the first evidence that a chaperonin molecule bears functionally
specialized surfaces for the binding of the protein substrates.
The term ``chaperonins'' denotes a family of ubiquitous, essential proteins endowed with ATPase activity that are involved in the folding, transport, and assembly of newly synthesized proteins (for reviews, see Ellis and van der Vies(1991), Hartl et al. (1992), and Horwich and Willison(1993)).
Eubacterial
chaperonins are structurally and functionally closely related to
eukaryotic-intraorganellar chaperonins. They have similar amino acid
sequences and consist of two stacked homo-oligomeric rings of seven
subunits of 60 kDa each that delimitate a central cavity;
sequence-related co-chaperonins, consisting of a single homo-oligomeric
ring of seven subunits of
10 kDa each, have been isolated together
with these chaperonins. The crystal structure determination of GroEL
(the Escherichia coli chaperonin) and a mutational analysis
revealed three domains in each subunit. The apical domain faces the
central cavity, and its flexible regions are involved in the binding of
the polypeptide substrate and the co-chaperonin GroES. The equatorial
domain provides most of the intratoroidal side-to-side contacts and all
of the ring-to-ring contacts and contains the ATP-binding site; the
domain intermediate between the other two probably allows allosteric
domain movement (Braig et al., 1994; Fenton et al.,
1994; Hartl et al., 1994). GroEL promotes correct folding by
binding to nonnative structures that are folding intermediates, thus
preventing the aggregation of hydrophobic surfaces, and coupling their
release in a correctly folded form to the binding and/or hydrolysis of
ATP; in some, but not all, cases the formation of native proteins
requires the presence of GroES.
Trent et al.(1991) reported that the chaperonin of the thermophilic archaeon Sulfolobus shibatae shows no significant relationship to GroEL-like chaperonins but has a 40% sequence identity with the chaperonins of the eukaryotic cytosol (named CCTs), reflecting the evolutive lineage of their sources. Archaeal chaperonins and CCTs do not have co-chaperonin equivalents; they show the typical double-ring structure with an 8- or 9-fold symmetry and a homo- or hetero-oligomeric structure. CCTs have a domain arrangement similar to that of GroEL; analysis of sequence relatedness to GroEL revealed a high level of identity in the regions corresponding to the ATP binding site, but no significant identity in the region corresponding to the polypeptide binding site (Kim et al., 1994; Kubota et al., 1994). In a functional regard, CCTs promote correct protein folding by an ATP-dependent mechanism similar to that exerted by GroEL but without the requirement for a co-chaperonin component (Frydman et al., 1992; Gao et al., 1992; Melki and Cowan, 1994).
The chaperonin of the
thermophilic archaeon S. solfataricus (Ssocpn), ()has a 9-fold symmetry (Knapp et al., 1994; Marco et al., 1994) and two subunit species that differ in size and
N-terminal sequences (Knapp et al., 1994), or one subunit of
60 kDa (Guagliardi et al., 1994; Marco et al.,
1994). Ssocpn displays a K
-dependent ATPase activity
and promotes correct refolding of several thermophilic and mesophilic
proteins in an ATP hydrolysis-dependent manner (Guagliardi et
al., 1994). In an effort to enlarge our knowledge about archaeal
chaperonins, we investigated the effects of Ssocpn on the aggregation
and inactivation upon heating of four model enzymes: chicken egg white
lysozyme (a monomer of 14.4 kDa), yeast
-glucosidase (a monomer of
68.5 kDa), chicken liver malic enzyme (a homotetramer of 65 kDa
subunits), and yeast alcohol dehydrogenase (a homotetramer of 37.5 kDa
subunits). This study provides, for the first time, biochemical
evidence that a chaperonin molecule possesses nonequivalent surfaces
for the binding of the protein substrates.
The assay
mixture for chicken egg white lysozyme consisted of 1 ml of a fresh
suspension 0.1 mg/ml of lyophilized E. coli cells in 50 mM Tris-HCl buffer, pH 7.4. After the addition of lysozyme, the time
required for an absorbance decrease of A = 0.1 was measured; the specific activity of the enzyme
was calculated by the formula time
mg
of protein according to Tsugita et al.(1968). Yeast
-glucosidase was assayed in 0.1 M sodium phosphate
buffer, pH 7.0, 0.3 mMp-nitrophenyl-
-D-maltoside (1 ml final volume)
according to Halvorson(1966); the continuous increase of absorbance at
400 nm was monitored, and an extinction coefficient for p-nitrophenol was 9.6 mM. Chicken liver ME was
assayed in 20 mM Tris-HCl buffer, pH 7.5, 0.05 mM NADP, 1 mM MgCl
, 1 mML-malate (1 ml final volume). Yeast ADH was assayed in 50
mM sodium phosphate buffer, pH 8.0, 2 mM NAD, 1
mM ethanol (1 ml final volume).
Figure 1:
Thermal aggregation
of chicken egg white lysozyme (upper panels) and yeast
-glucosidase (lowerpanels) in the absence (open symbols) and in the presence (closed symbols)
of Ssocpn. Lysozyme (80 µg in 1 ml) was incubated at 70 °C in
10 mM Tris-HCl buffer, pH 8.0, in the absence (
) or in
the presence (
) of Ssocpn at a molar ratio of 1:1 between the
chaperonin oligomer and the lysozyme chain.
-Glucosidase (200
µg in 1 ml) was incubated at 40 °C in 50 mM sodium
phosphate buffer, pH 8.0, in the absence (
) or in the presence
(
) of Ssocpn at a molar ratio of 1:1 between the chaperonin
oligomer and the
-glucosidase chain.
In a reasonable interpretation of the results,
Ssocpn did not rescue the protein aggregates, but rather it interacted
with lysozyme or -glucosidase before their precipitation and so
maintained them in solution. The hydrophobic surfaces of Ssocpn
contributed to the binding, as demonstrated by the finding that
aggregation occurred as in the absence of Ssocpn if the solutions of
lysozyme and
-glucosidase contained the chaperonin plus 0.1%
Triton X-100. The addition of ATP/Mg/K discharged the proteins that
proceeded to aggregate at the temperatures of the incubation. No
aggregation was detected if ATP/Mg (0.5 mM ATP, 0.5
mM MgCl
) was added instead of ATP/Mg/K; that is,
because Ssocpn displays a K
-dependent ATPase activity
(Guagliardi et al., 1994), ATP hydrolysis was required to
disrupt the binding between Ssocpn and the protein chain. Lysozyme and
-glucosidase discharged from Ssocpn after ATP hydrolysis
reaggregated, even though the test tube contained Ssocpn; in other
words, under the experimental conditions described, Ssocpn was not
viable for rebinding the denaturation intermediates. We are currently
investigating the recycling of Ssocpn by studying the effects of ATP
hydrolysis on its conformation, and the chaperonin affinity toward the
target protein, potassium ions, and ATP during in vitro functioning.
We determined the time courses of inactivations of
lysozyme and -glucosidase at the designated temperatures in the
absence and in the presence of Ssocpn oligomer at an equimolar ratio
with the protein chain (Fig. 2). Ssocpn did not modify the
kinetics of inactivation; that is, Ssocpn did not prevent the loss of
activity of the two enzymes. Following the addition of ATP/Mg/K (but
not of ATP/Mg), final activity regains of 78 and 55% were obtained in
the solutions of lysozyme and
-glucosidase heated in the presence
of Ssocpn. Lysozyme and
-glucosidase activities decreased when the
incubations were continued after the addition of ATP/Mg/K (not reported
in the figure), which is in accordance with the intrinsic heat lability
of the native enzymes. These results show that the molecules remained
in a folding-competent conformation while bound to the chaperonin and
that correctly folded molecules were released in solution upon ATP
hydrolysis.
Figure 2:
Thermal inactivations of chicken egg white
lysozyme (upper panels) and yeast -glucosidase (lower
panels) in the absence (open symbols) and in the presence (closed symbols) of Ssocpn. Lysozyme and
-glucosidase
were heated exactly as described in the legend to Fig. 1. At the
times defined, the enzyme activity was assayed as described under
``Experimental Procedures'' on aliquots drawn from each
mixture; the activity regains were calculated as percentages with
respect to the specific activities of the native enzymes. The scale of
the x axis changed at the time indicated by the arrow.
The final percents of active lysozyme or
-glucosidase did not vary when the chaperonin was present in the
solutions at a molar excess over the protein chain or when a molar
ratio of 1:2 between Ssocpn and the protein chain was used. At a molar
ratio of Ssocpn to protein chain of 1:10, lysozyme activity was
restored by 37% and
-glucosidase activity by 20%.
Figure 3:
Thermal aggregations of chicken liver ME (upper panels) and yeast ADH (lower panels) in the
absence (open symbols) and in the presence (closed
symbols) of Ssocpn. ME (6 µg in 1 ml) was incubated at 50
°C in 10 mM Tris-HCl buffer, pH 8.0, in the absence
() or in the presence (
) of Ssocpn at a molar ratio of 1:1
between the chaperonin oligomer and ME oligomer. ADH (6 µg in 1 ml)
was incubated at 50 °C in 10 mM Tris-HCl buffer, pH 8.0,
in the absence (
) or in the presence (
) of Ssocpn at a
molar ratio of 1:1 between the chaperonin oligomer and ADH
oligomer.
These results suggest that Ssocpn interacted with ME and ADH and prevented their precipitation upon heating; the hydrophobic nature of the interaction was demonstrated by the fact that 0.1% Triton X-100 in the solutions of ME and ADH containing Ssocpn abolished the protection against aggregation. The bound molecules were not released upon ATP binding or hydrolysis.
During heating, ME and ADH very rapidly lost their activity in the absence of Ssocpn but retained total activity in the presence of the chaperonin; the addition of ATP/Mg/K did not exert any effect (Fig. 4). We verified that the addition of Ssocpn to native solutions of ME and ADH did not affect their specific activities. In conclusion, the binding of ME or ADH to Ssocpn during heating did not cause any perturbation of the enzyme active sites, and the active conformations of the two enzymes were preserved.
Figure 4: Thermal inactivations of chicken liver ME (upper panels) and yeast ADH (lower panels) in the absence (open symbols) and in the presence (closed symbols) of Ssocpn. ME and ADH were heated exactly as described in the legend to Fig. 3. Enzyme activities were assayed as described under ``Experimental Procedures'' on aliquots drawn from each mixture.
No inactivation at all was detected when Ssocpn was incubated with ME or ADH at a molar ratio of 1:10; when Ssocpn and ME or ADH were heated at a molar ratio of 1:20, residual ME and ADH activities of about 30% were calculated after 30 min.
Figure 5:
A, solutions (0.3 ml) of lysozyme (10
µg), -glucosidase (20 µg), ME (30 µg) and ADH (30
µg) containing Ssocpn at an equimolar ratio with each protein
molecule were heated at the designated temperatures; after a 2-h
incubation, each solution was chromatographed on a Superose 6 column (1
48 cm; flow rate of 12 ml/h at 4 °C; eluent 10 mM
Tris-HCl, pH 8.4, 0.2 M NaCl). Left, the
chromatographic profiles relative to the runs: the elution volumes (ml)
are plotted against the absorption at 280 nm; the letters indicate the
protein used: a, lysozyme; b,
-glucosidase; c, ME; d, ADH. Right, the SDS-PAGE analysis
of the peaks from the column; the lanes were loaded with an aliquot
from the peak of the corresponding run. B, solutions of
lysozyme,
-glucosidase, ME, and ADH plus Ssocpn as in A were heated at the designated temperatures; after a 2-h
incubation, ATP/Mg/K was added to the solutions; 5 min after the
addition, each solution was chromatographed onto a Superose 6 column
(conditions were as described above). Axes and letters on the plots are
as in A; the peak of ATP was omitted. C, solutions of
lysozyme,
-glucosidase, ME and ADH plus Ssocpn as in A were directly chromatographed onto a Superose 6 column (conditions
were as above). Axes and letters on the plots are as in A.
Exploiting the same experimental tool, we
demonstrate that 1) the interaction of Ssocpn with the single-chain
proteins was reversed upon ATP hydrolysis, free lysozyme, or
-glucosidase being catalytically active and 2) the interaction of
Ssocpn with the tetrameric proteins was unaffected by ATP hydrolysis (Fig. 5B).
We demonstrate that Ssocpn does not interact with the native conformations of the model proteins (Fig. 5C). The chromatography on Superose 6 of nonheated solutions separated the protein used (eluted in an active form in a position corresponding to its native molecular weight) from Ssocpn (eluted in the void volume); SDS-PAGE analysis of the first eluting peak showed only the band corresponding to the Ssocpn subunit (not shown). Therefore, Ssocpn prevents the thermal aggregation of the model proteins by coating with its hydrophobic surfaces the interactive hydrophobic patches exposed on the intermediates of the thermal denaturation process.
To ascertain whether lysozyme and -glucosidase have
the same binding surfaces, we saturated Ssocpn with the denaturation
intermediate of
-glucosidase and tested if this chaperonin could
prevent the thermal aggregation of lysozyme. Ssocpn and
-glucosidase in a molar ratio of 1:50 were heated at 40 °C for
2 h; an ultracentrifugation at 50,000 rpm for 1 h pelletted visible
aggregates. Gel filtration of the supernatant on Superose 6 column (1
48 cm; eluent 10 mM Tris-HCl, pH 8.4, 0.2 M
NaCl; flow rate of 12 ml/h at 4 °C) yielded the
Ssocpn
-glucosidase complex (as judged by SDS-PAGE analysis)
in the void volume. We verified that all Ssocpn molecules in the peak
were saturated with
-glucosidase. In fact, the inclusion of a
protein excess from the peak in a solution of
-glucosidase heated
at 40 °C did not prevent the occurrence of aggregates (no
aggregation was detected when only the aliquot from the peak was
heated).
Ssocpn saturated with -glucosidase (briefly
Ssocpn/
-glucosidase) was included in a solution of lysozyme in a
protein excess over the lysozyme molecule, and the light scattering was
continuously monitored during heating at 70 °C, a light scattering
increase similar to that obtained when lysozyme was incubated in the
absence of the chaperonin was recorded (Ssocpn/
-glucosidase alone
did not aggregate over the time of the heating under the same
conditions). This result means that the thermal denaturation
intermediate of lysozyme did not find surfaces available for the
binding to Ssocpn/
-glucosidase. In a control experiment, we
included in the solution of lysozyme to be heated an
Ssocpn
-glucosidase complex, which was prepared at a molar
ratio of 1:0.5 between the chaperonin and the
-glucosidase chain
and was subjected to the same experimental protocol: in this case,
lysozyme aggregation was completely suppressed.
By the same
strategy, we demonstrated that the denaturation intermediates of ME and
ADH have the same binding surfaces on Ssocpn. We prepared a chaperonin
saturated with the thermal denaturation intermediate of ME by heating
at 50 °C for 1 h a solution in which the molar ratio between Ssocpn
and the substrate molecule was 1:80. The aggregates were removed by
ultracentrifugation, and the saturated form of Ssocpn (Ssocpn/ME unable
to prevent the aggregation of free ME) was recovered in the peak
eluting with the void volume from the Superose column loaded with the
supernatant (see above for details). Ssocpn/ME was unable to prevent
the aggregation at 50 °C of a solution of ADH; vice versa,
an SsocpnME complex, which was prepared at a molar ratio of 1:1
and was subjected to the same experimental protocol as that used to
prepare Ssocpn/ME, suppressed ADH aggregation
Surprisingly, when
Ssocpn/ME was included in solutions of lysozyme and -glucosidase
and the solutions were heated at the respective temperatures, no
aggregations were detected; active molecules were regained upon the
addition of ATP/Mg/K. When Ssocpn/
-glucosidase was included in
solutions of ME and ADH and the solutions were heated at 50 °C,
aggregations and inactivations were prevented. Since no artifact seemed
responsible for these results, Ssocpn has surfaces specific for the
binding of the denaturation intermediates of the single-chain proteins
(Surfaces I), which are not saturable by the thermal denaturation
intermediates of the tetrameric proteins; other surfaces specific for
the binding of the denaturation intermediates of the tetrameric
proteins (Surfaces II) are not saturable by the thermal denaturation
intermediates of the single-chain proteins. Consequently, it appears
that Ssocpn has nonequivalent surfaces for the binding of the model
proteins during their heating.
Some native proteins form visible aggregates when subjected to heating, as a consequence of hydrophobic intermolecular interactions; protein thermal aggregation is responsible for an irreversible loss of biological activity. We studied the effects of the chaperonin from the archaeon S. solfataricus, Ssocpn, on the in vitro thermal aggregation of two single-chain enzymes of 14.4 and 68.5 kDa and two tetrameric enzymes of 260 and 150 kDa.
Like other chaperonins, Ssocpn does not rescue proteins once they aggregate and does not bind the native molecules. Ssocpn prevents protein aggregation by interacting with the hydrophobic surfaces of the thermal denaturation intermediates of the model proteins, thus suppressing the intermolecular interactions that lead to aggregation. Because the tetrameric proteins retain their enzymatic activity upon binding to Ssocpn, the structure of the intermediate bound by Ssocpn is probably very similar to that of the native molecule. Ssocpn does not prevent the thermal denaturation of the single-chain enzymes that leads to their loss of activity, rather these enzymes are kept in a form that is refoldable upon ATP hydrolysis. As demonstrated for GroEL (Jackson et al., 1993; Hartl, 1994; Todd et al., 1994; Weissman et al., 1994), the binding and hydrolysis of ATP could trigger cycles of release from and rebinding to Ssocpn of the molecules until they have reached a correctly folded form.
The binding of the model proteins to Ssocpn during heating is not casual in the sense that the chaperonin surfaces are not indifferently available for the binding of any molecule; thermal denaturation intermediates of the single-chain proteins share Surfaces I, while thermal denaturation intermediates of the tetrameric proteins share Surfaces II.
There is
evidence that the binding of ATP to Ssocpn drives a conformational
change in the overall structure of the chaperonin (Guagliardi et
al., 1994; Knapp et al., 1994). To gain insight into the
functional consequence of this change, we heated lysozyme,
-glucosidase, ME, and ADH in the presence of the ATP-induced form
of Ssocpn and found that only the aggregations of the tetrameric
enzymes were suppressed, not the aggregations of the single-chain
enzymes, which proceeded as in the absence of the chaperonin (not
shown). Therefore, the ATP-induced form of Ssocpn does not bind the
thermal denaturation intermediates of the single-chain enzymes, but it
is still able to bind the thermal denaturation intermediates of the
tetrameric enzymes. This finding implies that ATP binding to Ssocpn
leads to a disappearance or modification of Surfaces I such that the
interaction with the proper molecules is prevented; Surfaces II are
probably less (or not at all) affected by the structural rearrangement.
It is noteworthy that the ATP-induced form of Ssocpn is unable to bind
the refolding intermediates of several chemically denatured proteins
(Guagliardi et al., 1994); we are currently investigating
whether the flexible Surfaces I are implicated also in the binding of
the interactive structures occurring during folding.
The domain organization of archaeal chaperonins is unknown. We suspect that Surfaces I could correspond to the flexible regions of the apical domain in GroEL involved in the binding of polypeptide substrate: these regions undergo a strong motion upon ATP-binding (Chen et al., 1994), and there is compelling evidence in the literature that the ATP-bound form of GroEL has a low affinity for unfolded protein substrates. It is unlikely that Surfaces II are located in the same region. The absence of any functional interference between the two Surfaces could imply that they are not in close vicinity on the chaperonin cylinder, and surfaces suited to bind molecules that exert catalytic activity could be surfaces exposed to the milieu; electron microscopy showed that the external envelope of GroEL binds the protein molecules (Azem et al., 1994). Our data show that Surfaces I and Surfaces II can bind more than one polypeptide chain during heating, which is consistent with the idea that chaperonins have multiple substrate binding sites. How many binding sites there are on Surfaces I and Surfaces II remains an open question, although it seems that the latter surfaces bind a higher number of substrate molecules than the former ones.
The activity of GroEL in preventing the
thermal aggregation of native proteins has been poorly investigated in
comparison with its activity in preventing the aggregation during
refolding of unfolded proteins. Upon heating in the presence of GroEL,
native -glucosidase (Holl-Neugebauer et al., 1991),
rhodanese (Mendoza et al., 1992), dihydrofolate reductase
(Martin et al., 1992), and malate dehydrogenase (Hartman et al., 1993) are protected against aggregation but not
against inactivation, and active molecules are released in solution
upon ATP hydrolysis. In an ATP-independent manner, GroEL keeps RNA
polymerase active (Ziemienowicz et al., 1993), interacts with
a conformation of 6-hydroxy-D-nicotine oxidase close to the
native state (Brandsch et al., 1992) and with a form of
rhodanese that is active (Mendoza et al., 1992).
Taken together, the aforementioned results about GroEL and the results of this study strongly suggest that chaperonins protect the native proteins against aggregative damage by two mechanistically distinct strategies. The cellular concentration of free ATP, which is high under physiological conditions and dramatically decreases upon different kinds of stress, may reflect the biological significance of the in vitro ATP-dependent strategy by which chaperonins prevent aggregation; thermal aggregation of proteins increases in ATP-depleted cells (Nguyen and Bensaude, 1994). The proteins that interact with the chaperonins in an ATP-independent fashion and in a conformation close to the native state probably undergo their cellular turnovers while bound to the chaperonin; active Ssocpn-bound ME and ADH lose their activity following trypsin digestion, and the inactivation profiles are identical to those of the free native molecules at the same protein concentrations (data not shown). In this scenario, the chaperonin-bound protein has the life-span of the free protein, which devised a strategy to avoid a sticky situation. The role of ``hydrophobic support'' played by the chaperonins in the cell is well suited to proteins that are very abundant also under physiological conditions, and, in terms of cell utility, one molecule that plays different roles via nonequivalent surfaces seems very advantageous.
Finally, we would like to underline an aspect of the research on chaperones from thermophilic Archaea. The protein named Hsp70 in Eukarya and DnaK in Eubacteria plays a central role in the heat shock response of these cells as a molecular chaperone involved in protein folding and transport; in E. coli, DnaK and related proteins cooperate with GroEL in a common pathway to facilitate protein folding (Gragerov et al., 1992; Hartl et al., 1994). Thermophilic Archaea do not possess an equivalent to DnaK, and chaperonins are the main proteins expressed under conditions of heat shock (Conway de Macario and Macario, 1994). Investigating the activity of archaeal chaperonins might strengthen the hypothesis that in Archaea chaperonins fulfill the functions of 70 kDa-chaperones in Eubacteria and Eukarya.