From the Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama, 226-8503, Japan
Received for publication, November 15, 2000, and in revised form, December 19, 2000
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ABSTRACT |
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Escherichia coli chaperonin
GroEL consists of two stacked rings of seven identical subunits each.
Accompanying binding of ATP and GroES to one ring of GroEL, that ring
undergoes a large en bloc domain movement, in which the
apical domain twists upward and outward. A mutant GroELAEX
(C138S,C458S,C519S,D83C,K327C) in the oxidized form is locked in a
closed conformation by an interdomain disulfide cross-link and cannot
hydrolyze ATP (Murai, N., Makino, Y., and Yoshida, M. (1996)
J. Biol. Chem. 271, 28229-28234). By reconstitution
of GroEL complex from subunits of both wild-type GroEL and oxidized
GroELAEX, hybrid GroEL complexes containing various numbers
of oxidized GroELAEX subunits were prepared. ATPase activity of the hybrid GroEL containing one or two oxidized
GroELAEX subunits per ring was about 70% higher than that
of wild-type GroEL. Based on the detailed analysis of the ATPase
activity, we concluded that inter-ring negative cooperativity was lost
in the hybrid GroEL, indicating that synchronized opening of the subunits in one ring is necessary for the negative cooperativity. Indeed, hybrid GroEL complex reconstituted from subunits of wild-type and GroEL mutant (D398A), which is ATPase-deficient but can undergo domain opening motion, retained the negative cooperativity of ATPase.
In contrast, the ability of GroEL to assist protein folding was
impaired by the presence of a single oxidized GroELAEX
subunit in a ring. Taken together, cooperative conformational
transitions in GroEL rings ensure the functional communication between
the two rings of GroEL.
The Escherichia coli chaperonin GroEL binds to
non-native proteins and facilitates their folding. GroEL is assisted by
the cofactor GroES and uses the energy of ATP hydrolysis (1, 2). GroEL
consists of fourteen identical 58-kDa subunits, which are arranged in
two heptameric rings stacked upon one another in a back to back manner,
forming a large central cavity (3-5). Each subunit of GroEL has a site
for ATP hydrolysis (4), and the ATPase cycle controls binding and
release of substrate protein, as well as GroES (6-8). ATP (or ADP)
induces a massive upward movement of the GroEL apical domains (5, 9),
which contain the binding sites for substrate proteins and for GroES
(10). Horovitz and coworkers (11) have proposed a nested model
describing the mixed cooperativity in ATP hydrolysis by GroEL, positive
cooperativity within the same rings and negative cooperativity between
the two rings. In the model, each GroEL ring is in equilibrium between two allosteric states, a tense (T) state with low affinity for ATP and
a relaxed (R) state with high affinity for ATP. Binding of ATP occurs
with strong positive cooperativity to one of the rings (12-15),
converting the T state to the R state (11). The allosteric transitions
of GroEL are most likely to correspond to the upward movement of the
apical domains (16). A second level of cooperativity undergoes
sequential transitions from the TT state via the TR state to
the RR state (11). This negative cooperativity between two rings
prevents ATP binding to the second ring (16).
We previously reported a mutant GroEL (GroELAEX;
C138S,C458S,C519S,D83C,K327C)1
in which apical and equatorial domains can be cross-linked in a
reversible manner (apical-equatorial cross
(X)-link) (17). Under reducing conditions,
GroELAEX is fully active as a chaperonin. However, under
oxidative conditions, a disulfide cross-link is formed between two
introduced cysteines, locking GroELAEX in a closed
conformation, which can bind ATP and polypeptide but is unable to
hydrolyze bound ATP, release bound polypeptide, or bind GroES (17).
Here, to investigate the role of each subunit in the tetradecameric
GroEL complex, we prepared intersubunit hybrid complexes containing
various copies of the oxidized GroELAEX monomers by
reconstituting the denatured GroEL monomers. Remarkably, the hybrid
GroEL complex containing one or two disulfide cross-linked GroELAEX monomers per ring showed larger ATPase activity at
saturating ATP concentration than wild-type GroEL
(GroELWT). This is likely caused by removal of the negative
cooperativity between rings. In contrast, hybrid between
GroELWT and GroELD398A, which is
ATPase-deficient but can undergo domain-opening motion, retained
negative cooperativity of the ATPase activity. The coordinate closed to
open conformational transition might be essential for the cooperative
nature of GroEL.
Materials--
GroELWT and
GroELAEX were purified from E. coli strain
BL21(DE3) bearing the plasmids pET-EL (18) and pT7AEX (17),
respectively, as described previously. The single-stranded DNA of the
plasmid pET-EL was obtained by infecting E. coli CJ236 cells
with helper phage M13KO7 (Amersham Pharmacia Biotech). Mutant GroEL,
GroELD398A, and GroELD398A/D490C were
generated by site-directed mutagenesis using the Kunkel method.
GroELD398A and GroELD398A/D490C were purified
through the similar protocol described for GroELWT (18). Purified GroELWT and its mutants were stored as a
suspension in 65% ammonium sulfate at 4 °C until use.
Isopropylmalate dehydrogenase (IPMDH) from Thermus
thermophilus strain HB8 was kind gift from Dr. T. Oshima (Tokyo
University of Pharmacy and Life Science, Hachioji, Japan).
(2R*,3S*)-3-Isopropylmalic acid, a substrate of IPMDH, was purchased
from Wako Chemical Co. (Osaka, Japan). All other chemicals were the
highest grade commercially available.
Preparation of Hybrid GroEL Tetradecamers--
The mixture of
GroELWT tetradecamer and mutant GroEL tetradecamer in
various ratios (total 10 mg/ml protein concentration) was denatured by
addition of urea (final concentration 8 M) at 25 °C for
90 min. Reconstitution to tetradecamers was accomplished by modified
protocol described in Ref 19. The urea-denatured GroEL was diluted
15-fold into buffer containing 50 mM Tris-HCl (pH 7.5)
followed by addition of 10 mM MgCl2, 5 mM ATP, and 0.6 M ammonium sulfate.
Reconstitution was allowed to continue for 30 min at 25 °C. The
reconstitution yield of the tetradecamer from the GroEL mutant subunits
(GroELAEX and GroELD398A) was as efficient as
that of GroELWT, reaching to 90% (data not shown). The
reconstituted GroEL was purified by using a gel filtration high
pressure liquid chromatography column (G3000SWXL; Tosoh) equilibrated with 25 mM Tris-HCl (pH 6.8) and 100 mM Na2SO4. The fractions containing
the tetradecamer were collected and concentrated with ultrafiltration
membrane (Ultrafree 10-kDa cut-off; Millipore). As we could distinguish
the GroELAEX complex from GroELWT complex in
native PAGE, random incorporation of GroELAEX subunits into the hybrid complex was also confirmed by native PAGE. Random
incorporation of GroELD398A subunits into the corresponding
hybrid complex was confirmed as follows. We replaced Asp-490, which is
located on the outer surface of GroEL, to Cys, in addition to the D398A
mutation (GroELD398A/D490C). The chemical modification of
this surface-exposed Cys could invest the complex with a different
electrophoretic mobility. The D490C mutation itself has no additional
effect on the property of the GroELD398A complex. After the
isolation of the hybrid complex formed between
GroELD398A/D490C and GroELWT, the introduced
Cys was chemically modified with (2-bromoethyl)trimethylammonium. Finally we could confirm the formation of the various hybrid complexes by native PAGE analysis (data not shown).
ATPase Assay--
ATPase activity was assayed using malachite
green to measure the amount of produced inorganic phosphate (20). The
assay solution was preincubated for 10 min at 25 °C and then
reaction was started by the addition of ATP (final concentration, 2 mM) to the assay solution containing 50 mM
Tris-HCl, pH 7.5, 5 mM MgCl2, 50 mM
KCl, 0.5 µM GroEL, unless otherwise stated. Assay solutions were incubated at 25 °C, and the reaction was terminated at several time points by addition of perchloric acid. The mixture was
then reacted with a malachite green reagent, and absorbance at 630 nm
was measured. One unit of activity is defined as the activity that
hydrolyzes 1 µmol of ATP/min. Data were fit to the equation for the
nested allosteric model developed by Horovitz and coworkers (11).
Folding Assay--
IPMDH from T. thermophilus (0.33 mg/ml) was denatured in 6.4 M guanidine HCl and diluted
50-fold into the dilution buffer (100 mM potassium
phosphate, pH 7.8, 1 mM MgCl2) containing, when indicated, 2.0 mM ATP and 0.19 µM GroEL. The
dilution buffer was preincubated for 10 min at 37 °C prior to
addition of denatured IPMDH. The mixtures were incubated 60 min at
37 °C, and an aliquot was injected into the assay solution, which
contained 100 mM potassium phosphate, pH 7.8, 1 mM MgCl2, 1.0 M KCl, 0.9 mM NAD+, 0.4 mM 3-isopropylmalic
acid. The increasing rate of absorbance at 340 nm was monitored at
60 °C, and the activity of recovered IPMDH was normalized relative
to that of IPMDH recovered by GroELWT.
Other Methods--
Proteins were analyzed by PAGE on a
polyacrylamide gel (13% (w/v) in the presence of 0.1% (w/v) SDS or
6% (w/v) without SDS). 2 µg of proteins were loaded on each lane of
the gels, and the reducing reagent was always omitted from the sample
solutions, the running buffer, and gels. Protein bands were visualized
by Coomassie Brilliant Blue R-250 staining. Protein concentrations were
assayed by the Bradford method with bovine serum albumin as a standard
(21). Throughout this study, the concentrations of GroEL are expressed
as tetradecamers.
Preparation of the Hybrid Complex between Wild-type GroEL and
Oxidized GroELAEX--
To prepare the intersubunit hybrid
complexes between GroELWT and oxidized
GroELAEX, we reconstituted GroEL tetradecamers from the
urea-denatured monomers in the presence of ATP and ammonium sulfate
(19) with various ratios of GroELAEX to GroELWT
(Fig. 1A). Regardless of the
contents of GroELAEX, the reconstitution was very
efficient, and more than 90% of the GroEL subunits were incorporated
into the complex. Tetradecameric structure of the reconstituted hybrids
was confirmed by gel filtration high pressure liquid chromatography
(data not shown). After isolation of the reconstituted hybrid complex
(termed HybridWT-AEX) by gel filtration, the formation of
the hybrid complex was confirmed with native PAGE (Fig. 1B).
In native PAGE, the oxidized GroELAEX tetradecamer migrated
faster than GroELWT (Fig. 1B). The
HybridWT-AEX appeared as smeared bands between them,
representing a mixture of GroEL hybrids containing various ratios of
oxidized GroELAEX and GroELWT. This indicated
that the formation of the hybrids occurred randomly in each of the
reassembled hybrid GroEL molecules in the solution. These isolated
hybrid GroEL tetradecamers did not reassemble to the parent GroEL after
storage at 4 °C for 1 week.
ATPase Activity of the Hybrids between GroELWT and
Oxidized GroELAEX--
Oxidized GroELAEX can
bind ATP but cannot hydrolyze it (17), so that ATPase activities of the
mixture of parent GroEL tetradecamers, i.e.
oxidized GroELAEX and GroELWT, are proportional
to the contents of GroELWT (Fig.
2A). In contrast, ATPase
activities of the reconstituted HybridWT-AEX with various
amounts of GroELAEX present were not proportional to the
amounts of oxidized GroELAEX. They showed almost
proportional activity until the ratio of WT:AEX was 1:6 but showed
higher activity than the proportional one when the ratio of WT:AEX was
between ~2:5 and 6.5:0.5. The ATPase activity of the hybrid complex
was at the maximum when the ratio of WT:AEX was 5:2. This result was
unexpected, because it has been believed that ATP hydrolysis by GroEL
is highly cooperative within each GroEL ring (11, 12), predicting that
incorporation of a single inactive subunit into the ring may be
sufficient to abolish the entire ATPase activity.
Dependence of ATPase Activity on ATP Concentration--
The ATPase
activity of GroELWT exhibits an apparent substrate
inhibition, which has been explained by negative cooperativity between
two rings (11). As the ATP concentration increases, ATPase activity of
GroELWT also increases, reaching the maximum activity at
0.1 mM ATP and then decreasing to about 65% of the maximum
value (Fig. 2C, closed circles). However, ATPase
activities of the 5:2 HybridWT-AEX, which has 1.6-fold
higher ATPase activity than that of GroELWT, did not show
the substrate inhibition (Fig. 2C, crosses). The
inhibition of ATPase at ATP concentrations above 0.1 mM ATP
is caused by the negative cooperativity between rings; at low ATP
concentrations, one of two rings of GroEL is involved in ATP hydrolysis
at a given moment, but at concentrations >0.1 mM ATP, the
other ring also binds ATP, resulting in inhibition of ATP hydrolysis of
the first ring. According to this scenario, HybridWT-AEX is
likely impaired in communication between rings because of the presence
of the GroELAEX subunits, which are locked in closed conformation.
Hybrid GroEL between GroELD398A and
GroELWT--
It is likely that the presence of one or two
closed GroEL subunits in the same ring prevents the suppressive
interaction between rings, and uninhibited ATPase activity occurs in
the wild-type subunits. Regarding this hypothesis, another
ATPase-deficient mutant, GroELD398A, was used to
investigate the role of conformational change in ATPase activity of
hybrid GroEL. GroELD398A is mostly deficient in the ATPase
activity because of the lack of carboxyl group required for the ATP
hydrolysis but can undergo domain-opening motion (7). Hybrid GroEL
composed of GroELD398A and GroELWT (termed
HybridWT-398) were prepared in a similar procedure as that
described for the HybridWT-AEX. Random incorporation of the GroEL subunits into the HybridWT-398 was confirmed by
native-PAGE (data not shown; see "Experimental Procedures"). ATPase
activity of the HybridWT-398 was nearly proportional to the
content of GroELWT (Fig. 2B), indicating that
the incorporated ATPase-deficient GroELD398A subunit did
not interfere the ATP hydrolysis of competent GroELWT
subunits. This result is in contrast to that of the
HybridWT-AEX, suggesting that the HybridWT-398
retained negative cooperativity of ATPase function. Indeed, dependence
of ATPase activity of the 5:2 HybridWT-398 on ATP
concentration showed substrate inhibition derived from the negative
cooperativity of ATPase even though the specific activity of the
complex was lower than that of GroELWT tetradecamer (Fig.
2D).
Chaperone Activity of Hybrid GroEL--
Chaperone activities of
the hybrid GroEL complexes were examined using guanidine HCl-denatured
IPMDH (22), which has no cysteine (23). All of the GroEL complexes
used, i.e. GroELWT, GroELAEX, GroELD398A, HybridWT-AEX,
and HybridWT-398, were able to bind denatured IPMDH and
arrest the spontaneous folding of IPMDH (data not shown). When ATP was
added, GroELWT released the arrested IPMDH into the medium
where IPMDH accomplished folding, and enzymatic activity was recovered.
The yield of recovered IPMDH thus measured for HybridWT-AEX
and mixtures of parent GroEL tetradecamers are shown in Fig.
2E. In contrast with the ATPase activity, chaperone activity
was severely inhibited by the presence of the oxidized GroELAEX subunits in the hybrid complex independent from
the number of GroELAEX. This result indicates that
incorporation of oxidized GroELAEX subunit of the
heptameric ring is sufficient to inhibit the chaperone activity.
Although the suppression of the chaperone activity was not complete,
the result clearly shows that the negative cooperativity between two
rings transmitted by conformational changes of the subunits is
important for the ability to release the non-native proteins.
In contrast to the HybridWT-AEX, the chaperone activity of
the HybridWT-398 was similar to that of mixture of parent
GroEL tetradecamers, indicating that there is no additional inhibitory effect of GroELD398A subunits in the
HybridWT-398 (Fig. 2F).
Importance of Cooperative Conformational Transition in GroEL
Rings--
The most compelling result in this report is that the
incorporation of even one or two cross-linked GroELAEX
mutants into the GroEL ring results in the stimulation in the ATPase
activity, but it suppresses the chaperone activity. Dependence of the
ATPase activity of the HybridWT-AEX on ATP concentration
reveals that there is no apparent substrate inhibition, which can be
explained by a disruption of the inter-ring negative cooperativity with respect to ATP. This indicates that the double rings of the
HybridWT-AEX forms the RR state upon ATP binding.
Disruption of the negative cooperativity has been also observed in the
GroEL (R13G,A126V) double mutant (24), which was used for determination
of the GroEL crystal structure (3, 4). Notably, the crystal structure with bound nucleotides has revealed that the double mutant unusually bound 14 ATP
Asymmetric behavior of GroEL double rings upon ATP binding
is very important in the chaperone function. Indeed, alternation of
active rings during the ATPase cycle is essential for the productive GroEL-promoted folding (6-8). It is plausible that release of substrate protein was inhibited in the HybridWT-AEX,
because the hybrid GroEL tetradecamer remains symmetric upon ATP
binding. Taken together, these results suggest that cooperative
conformational transitions within one GroEL ring ensure proper
communication between the two rings, which are necessary for
maintenance of the asymmetric GroEL reaction cycle.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Preparation of intersubunit hybrid between
GroELWT and GroELAEX. A,
schematic illustration of procedures to prepare the hybrid GroEL
complexes. Urea-denatured GroEL, wild-type and mutant, was mixed at
various ratios and then diluted into the buffer containing ATP,
MgCl2, and ammonium sulfate. After the incubation at
25 °C for 30 min, the reconstituted hybrid GroEL complexes were
purified by gel filtration high pressure liquid chromatography.
B, native PAGE analysis of the isolated
HybridWT-AEX. Isolated HybridWT-AEX prepared in
indicated molar ratios were analyzed by native PAGE. ±, +/+, and /+
indicate the marker for the parent GroEL complexes, GroEL wild-type
(WT), and oxidized GroELAEX
(AEX).
View larger version (27K):
[in a new window]
Fig. 2.
Properties of hybrid GroEL, the
HybridWT-AEX, and the HybridWT-398.
The left three panels (A, C, and
E) contain the HybridWT-AEX; right three
panels (B, D, and F) contain the
HybridWT-398. A and B, ATPase
activities of hybrid GroEL complexes in various ratios.
mixture indicates the minimal mixtures of parent GroEL
complexes in the indicated ratios. Assays were carried out at 25 °C
for 10 min. Activity of GroELWT, 0.036 µmol/mg/min, was
set as 100%. Arrows indicate the activities of 5:2
(wild-type:mutant) hybrid GroEL. C and D, effect
of ATP concentration on the ATP hydrolysis by the hybrid GroEL. 5:2
hybrid GroEL (80 nM), indicated by arrows in
A and B, was subjected to the ATPase assay for 3 min. As a control, results obtained from wild-type GroEL
(WT) are also shown. Data were fit to the equation for the
nested allosteric model (11). E and F, effect of
hybrid GroEL tetradecamers on folding of IPMDH. IPMDH denatured in 6.4 M guanidine HCl was diluted 50-fold into the buffer
containing the hybrid GroEL tetradecamers at 37 °C and then followed
by the addition of ATP. After a 60-min incubation, recovered IPMDH
activity was measured. The extent of recovered IPMDH activity was
expressed by the activity recovered by GroELWT. Under these
conditions, ~70% of denatured IPMDH spontaneously,
i.e. without GroEL and ATP, folds.
S per tetradecamer (4), suggesting that a symmetric RR
state was achieved upon ATP binding (24). The structural basis for the
disruption of the negative cooperativity in the double mutant is not
clear. In contrast, disruption of the negative cooperativity in the
HybridWT-AEX is because of its inability to undergo the
required conformational change in the incorporated GroELAEX
subunits. On the other hand, the hybrid GroEL complex containing
GroELD398A, which can undergo domain-opening motion (7),
retained the negative cooperativity like wild-type GroEL. We therefore
conclude that the negative cooperativity between rings requires a
coordination of cooperative conformational transition within rings.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jeanne Hardy for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by a grant-in-aid for Scientific Research on Priority Areas (A) from the Ministry of Education, Science, Sports and Culture of Japan.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.
Present address: Dept. of Biochemistry 2, Jikei University School
of Medicine, 3-25-8, Nishishinnbashi, Minato-ku, Tokyo 105-8461, Japan.
§
To whom correspondence should be addressed: Chemical Resources
Laboratory, R1, Tokyo Inst. of Technology, 4259 Nagatsuta, Yokohama,
226-8503, Japan. Tel.: 81-45-924-5232; Fax: 81-45-924-5277; E-mail:
htaguchi@res.titech.ac.jp.
Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M010348200
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ABBREVIATIONS |
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The abbreviations used are:
GroELAEX, GroEL mutant (C138S, C458S,C519S,D83C,K327C);
GroELWT, wild-type GroEL;
GroELD398A, GroEL
mutant (D398A);
HybridWT-AEX, intersubunit hybrid GroEL
tetradecamer between GroELWT and
GroELAEX;
HybridWT-398, intersubunit hybrid
GroEL tetradecamer between GroELWT and
GroELD398A;
IPMDH, isopropylmalate dehydrogenase from
T. thermophilus;
PAGE, polyacrylamide gel electrophoresis;
ATPS, adenosine 5'-O- (thiotriphosphate);
GroELD398A/D490C, GroEL mutant
(D398A,D490C).
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REFERENCES |
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