Discrimination of ATP, ADP, and AMPPNP by Chaperonin GroEL
HEXOKINASE TREATMENT REVEALED THE EXCLUSIVE ROLE OF ATP*
Fumihiro Motojima and
Masasuke Yoshida
From the
Chemical Resources Laboratory, Tokyo Institute of Technology, 4259
Nagatsuta, Midori-ku, Yokohama 226-8503, Japan
Received for publication, January 24, 2003
, and in revised form, May 7, 2003.
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ABSTRACT
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The double ring chaperonin GroEL binds unfolded protein, ATP, and GroES to
the same ring, generating the cis ternary complex in which folding
occurs within the cavity capped by GroES (cis folding). The
functional role of ATP, however, remains unclear since several reports have
indicated that ADP and AMPPNP
(5'-adenylyl-
,
-imidodiphosphate) are also able to support
the formation of the cis ternary complex and the cis
folding. To minimize the effect of contaminated ATP and adenylate kinase, we
have included hexokinase plus glucose in the reaction mixtures and obtained
new results. In ADP and AMPPNP, GroES bound quickly to GroEL but bound very
slowly to the GroEL loaded with unfolded rhodanese or malate dehydrogenase.
ADP was unable to support the formation of cis ternary complex and
cis folding. AMPPNP supported cis folding of malate
dehydrogenase to some extent but not cis folding of rhodanese. In the
absence of hexokinase, apparent cis folding of rhodanese and malate
dehydrogenase was observed in ADP and AMPPNP. Thus, the exclusive role of ATP
in generation of the cis ternary complex is now evident.
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INTRODUCTION
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The bacterial chaperonin system consisting of GroEL and GroES facilitates
folding of other proteins using the energy of ATP hydrolysis. GroEL is
composed of 14 identical 57-kDa subunits, each containing a site for binding
and hydrolysis of ATP. Seven GroEL subunits are arranged in a heptamer ring
forming a central cavity, and two heptamer rings are stacked back to back.
GroES is a dome-shaped, single heptamer ring of 10-kDa subunits. GroEL binds a
wide range of unfolded proteins at the apical cavity surface and subsequently
binds ATP and GroES to the same GroEL ring (the cis ring, a GroEL
heptamer ring that binds to GroES), producing the complex consisting of GroEL,
unfolded protein, and GroES (the cis ternary complex). Since the
residues of the GroEL apical surface involved in GroES binding are mostly
overlapped with substrate protein binding
(1), GroES binding results in
encapsulating unfolded protein into the enlarged cavity of the cis
GroEL ring capped by GroES (the cis cavity)
(2). The unfolded protein
initiates folding in the cis cavity without a risk of aggregation
(the cis folding). ATP hydrolysis in the cis ring and
subsequent ATP binding to the opposite side of GroEL ring (the trans
ring) induce the release of GroES, ADP, and substrate protein (whether folded
or not) from the cis ring
(3,
4). When unfolded protein is
added to the GroEL-GroES complex, it binds to the trans ring, and its
folding is arrested (2).
Binding and release from the trans ring enable the folding for some
proteins (2), especially large
ones that are too large to be encapsulated in the cis cavity, by
lowering the concentration of aggregation-prone folding intermediates in bulk
solution (5). In contrast, the
stringent substrate proteins for chaperonin fold efficiently by the
cis folding in the presence of ATP
(3,
6).
However, it should be noted that the functional significance of ATP for the
cis ternary complex formation is unclear yet. It was reported that
slow cis folding of rhodanese, a stringent substrate protein, is
observed when GroES and ADP or
AMPPNP1 were added to
GroEL-unfolded rhodanese complex
(2,
7,
8). The cis folding of
dehydrofolate reductase in the presence of ADP was also reported
(9). Malate dehydrogenase (MDH)
and Rubisco were shown to form the cis ternary complex in ADP.
However, these complexes did not promote folding
(3). These observations have
raised an intriguing question: what is the difference between the
non-productive ADP-induced cis ternary complex and the productive
ATP-induced cis ternary complex?
As reported previously
(10), we have noticed that
inclusion of hexokinase and glucose in the reaction mixtures diminished
ADP-dependent cis folding of green fluorescent protein. Commercially
prepared ADP and AMPPNP usually contain a trace amount of ATP. Also, if a
trace amount of adenylate kinase is contaminated in purified protein, it can
produce ATP from ADP constantly. Hexokinase would eliminate these unwanted
ATPs during the reaction period. Another potential factor that can affect the
results is heterogeneity of GroEL complexes in the reaction mixtures. For
example, if two GroEL rings are not saturated by unfolded proteins and if
GroES can bind preferably to free GroEL rings rather than the unfolded
protein-loaded GroEL rings, two kinds of complexes would be generated:
GroEL-unfolded protein complex without GroES or GroEL-GroES complex without
unfolded protein in the cis cavity. Since these two complexes cannot
be separated, the results may be taken as an evidence for the existence of the
GroEL-GroES-unfolded protein ternary complex. Here, we reexamined the
nucleotide requirement for the cis ternary complex by taking the
precautions described above. The results showed that ADP is incompetent to
generate the cis ternary complex, that is, ATP is stringent for the
fast GroES binding to the unfolded protein-loaded GroEL rings and subsequent
cis folding of substrate protein.
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MATERIALS AND METHODS
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MaterialsSingle-stranded DNAs of the plasmid pET-EL and
pET-ES2 were obtained by infecting Escherichia coli CJ236 cells with
helper phage M13KO7 (Amersham Biosciences). Mutant GroES(T19C) was made by
Kunkel methods using an oligonucleotide
5'-CAGCAGATTTGCATTCAACTTCTTTACG-3'. GroEL, GroES mutants, and
rhodanese were expressed and purified as described
(11). GroEL purified by the
procedures including gel permeation column chromatography in the presence of
30% methanol (11) contained
only a very small amount of contaminated proteins (<0.1 Trp residues/GroEL
tetradecamer). MDH from pig heart, hexokinase, ATP, ADP, and AMPPNP were
purchased from Roche Diagnostics. Diadenosine pentaphosphate (Ap5A) was
purchased from Sigma. Protein concentrations were measured by BCA protein
assay (Pierce) and calibrated on the basis of quantitative amino acid
analysis.
GroEL Saturated with Unfolded ProteinsRhodanese (4
µM) or MDH (4 µM) was heat-denatured at 60 °C
for 15 min in the buffer (50 mM HEPES-NaOH, pH 7.2, 1 mM
EDTA, and 1 mM DTT) containing 1 µM
GroEL.2 By this
treatment, GroEL was not impaired at all, as shown by its 100% retention of
ATPase activity and chaperone activity, whereas substrate proteins were
completely inactivated. Temperature was shifted down to 25 °C, and
GroEL-unfolded protein complex was isolated with gel permeation HPLC. In the
latter stage of this study, we used ultrafiltration (Microcon YM-100,
Millipore), which was as effective as HPLC to remove unbound substrate
proteins.
cis Folding AssaysTo initiate cis folding
reactions, the solution containing GroEL loaded with unfolded proteins
(rhodanese or MDH) and GroES was mixed with a 3-fold volume of the solution
containing the nucleotide (ATP, ADP, or AMPPNP). When indicated, hexokinase
was included in the nucleotide (ADP or AMPPNP) solutions. Final concentrations
of the components in the reaction mixtures were 1 mM nucleotides,
0.5 µM GroEL saturated with rhodanese or MDH, 1.0
µM GroES, 200 mM glucose, 50 mM
HEPES-NaOH, pH 7.2, 200 mM CH3COOK, 10 mM
Mg(CH3COO)2, 1 mM DTT and, when indicated,
0.04 units/µl hexokinase. In the case of rhodanese, 20 mM
Na2S2O3 was additionally included in the
reaction mixtures. For the single turnover ATP hydrolysis experiment, excess
ATP was hydrolyzed by adding hexokinase (final concentration, 0.04
units/µl) to the reaction mixture at 3 s after initiation of the reaction.
We confirmed that 1 mM ATP in the reaction mixture was quenched
completely within next 3 s. To measure the time course of rhodanese folding,
aliquots (5 µl) were taken out at indicated times and mixed with 750 µl
of the solution containing 100 mM KH2PO4, 150
mM Na2S2O3, and 1 mM
EDTA, and recovered rhodanese activities were measured
(8,
11). In the experiments to
observe the effect of pretreatment with hexokinase, the nucleotide solutions
were treated with hexokinase (0.04 units/µl) for 15 min and separated from
hexokinase by ultrafiltration (Microcon YM-3). When indicated, 1 mM
Ap5A was included in the nucleotide solutions. The abovementioned assays were
all carried out at 25 °C. To quantitate the amount of GroES and the
substrate protein in the cis cavity, at 2 h of incubation, released
GroES and substrate protein were removed by repeated (four times)
dilution-ultrafiltration procedures (Microcon YM-100). Subsequently,
proteinase K (final concentration, 1 µg/ml) was added to digest
non-protective unfolded protein. After a 30-min incubation at 25 °C,
phenylmethylsulfonyl fluoride was added at final concentration of 1
mM, and digested products smaller than 100 kDa were removed by
ultrafiltration as described above. An aliquot of the resulting solution
(
20 µg) was applied to 12% polyacrylamide gel electrophoresis in the
presence of SDS, and Coomassie Blue R-250-stained band intensities of
substrate proteins and GroES were quantitated by using the public domain NIH
Image program (developed at the U.S. National Institutes of Health and
available on the Internet at
rsb.info.nih.gov/nih-image)
and calibrating with band intensities of the known amount of proteins.
Additional aliquots of proteinase K-treated, ultrafiltered samples were used
for measuring the recovered activities of rhodanese or MDH that corresponded
to the cis folding. To release all the proteins from the cis
cavity, GroES was detached from GroEL by the addition of an equal volume of 50
mM EDTA to the aliquot. After a 90-min incubation at 25 °C (in
which active dimers were formed in the case of MDH), activities of rhodanese
and MDH were measured.
Kinetics of GroES Binding to and Release from
GroELGroES(98C) and GroES(T19C) (0.14 mM) were
incubated at 4 °C for 24 h with 2 mM 5(2-iodoacetylaminoethyl)
aminonaphthalene-1-sulfonic acid in 25 mM Tris-HCl, pH 7.5, and 10
mM Tris(2-carboxyethyl)phosphine (Molecular Probes). Free labeling
reagent was quenched by 10 mM DTT and removed by a Sephadex G-25
column equilibrated with 50 mM HEPES-NaOH, pH 7.2, 200
mM CH3COOK, 10 mM
Mg(CH3COO)2, and 1 mM DTT. The fractions
containing GroES were concentrated by Ultrafree-4 (Millipore). The molar ratio
of the labels to GroES was
0.2:1 for both GroESs. The labeled GroES(98C)
(GroESC) and labeled GroES(T19C) (GroESN) were fully
active in GroEL-GroES-dependent folding of rhodanese and in suppression of
ATPase activity of GroEL (data not shown). The time course of GroES binding to
GroEL was monitored by the fluorescence of GroESN that increased
upon binding to GroEL. The solution containing GroESN and
nucleotide was mixed at 25 °C with free GroEL or GroEL saturated with
unfolded proteins, prepared as described above, using a stopped-flow rapid
mixing apparatus (SFM-400, BioLogic) equipped with fluorometer. Final
concentrations of GroESN and nucleotide were 0.02 µM
and 1 mM, respectively. GroEL concentrations were varied (final
concentrations, 0.05, 0.1, 0.15, 0.2, and 0.25 µM). The
excitation wavelength was 340 nm, and the emission light above 420 nm was
measured. The same experiments were repeated five times, and the data were
averaged. The binding was analyzed assuming the simple bimolecular binding
scheme, GroEL + GroESN
GroEL-GroESN. Since the
dissociation rate constant (koff) is small enough to be
ignored, the association rate constant kon is calculated
from linear fitting to the apparent rate constants (kapp)
obtained from single exponential fitting at different GroEL concentrations;
kapp = kon [GroEL]. The rate of
release of GroES from GroEL was estimated from the loss of fluorescence of
GroESC from the GroEL-GroESC complexes. The cis
ternary GroEL-GroESC-rhodanese complex and the
GroEL-GroESC complexes were made by the procedures described above
except that 1 µM GroEL and 0.5 µM
GroESC were used in the presence or absence of rhodanese. The
complexes were incubated at 25 °C with excess amount (final concentration,
10 µM) of non-fluorescent, native GroES to prevent the rebinding
of GroESC. Aliquots were taken out at indicated times and applied
to a gel permeation HPLC column equilibrated with 50 mM HEPES-NaOH,
pH 7.2, 200 mM CH3COOK, 10 mM
Mg(CH3COO)2, 0.1 mM ADP, 20 mM
glucose, 0.01 units/µl hexokinase, 1 mM DTT, and 0.05%
NaN3. The GroESC fluorescence eluted in the GroEL peak
was measured with an on-line fluorometer at 490 nm with excitation at 340
nm.
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RESULTS AND DISCUSSION
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Elimination of Contaminated ATP by HexokinaseTrace amount
of ATP in ADP and AMPPNP solutions cannot be accurately measured with the
usual HPLC analysis, and we applied a luciferase assay. Commercially available
ADP and AMPPNP usually are contaminated with ATP, ranging 0.12.0%.
Purification of ADP using a Dowex ion exchange column decreased the amount of
ATP to 0.020.05%. Treatment with hexokinase plus glucose was more
effective, and ATP contamination in ADP and AMPPNP was decreased down to
0.0050.009% (10).
Taking into account that purified protein could contain trace amounts of
adenylate kinase that potentially generate ATP (and AMP) from ADP continuously
during the reaction period, we have included hexokinase plus glucose in the
reaction mixtures. Hereafter, commercial AMPPNP and ADP that have not been
treated with hexokinase are referred to as AMPPNPraw and
ADPraw, respectively. Those treated with hexokinase plus glucose
are as AMPPNPhex and ADPhex, respectively. Hexokinase
was also used for the single turnover ATP hydrolysis experiment (referred to
as ATPsingle); hexokinase added at 3 s after initiation of the
reaction hydrolyzed all free ATP so that the second chaperonin cycle was
prevented. The folding yields of rhodanese and MDH in the cis cavity
under the conditions of ATPsingle were 1.1 and 0.60 mol/mol of
GroEL-GroES complex, respectively, which were approximately the same as those
(1.2 and 0.66 mol/mol) achieved by GroEL(D398A), a mutant deficient in ATP
hydrolytic activity. Therefore, it is confirmed that the single turnover
reaction of chaperonin can be measured by this procedure.
Saturation of GroEL by Heat-denatured ProteinsAt first, we
prepared GroEL in which all the binding sites for substrate proteins were
saturated with unfolded proteins. Dilution from the concentrated protein
solutions containing chemical denaturants such as guanidium hydrochloride or
urea into the reaction solution was avoided because of the inhibition effect
of the denaturants on GroEL-GroES association
(12). Instead, we simply
heated the mixtures of GroEL and substrate proteins, either rhodanese or MDH,
at 60 °C for 15 min. Rhodanese and MDH were denatured completely by this
heat treatment. GroEL is a rather heat-stable protein and remained intact as
ensured by the unaffected ATP hydrolysis activity and chaperone activity after
the heat treatment (data not shown). Denatured substrate proteins occupied all
the substrate protein binding sites of GroEL, and the GroEL loaded with a
saturating amount of unfolded proteins (referred as the loaded GroEL) was
separated from unbound substrate proteins with gel permeation HPLC or
ultrafiltration. The molar stoichiometry of the bound substrate protein to
GroEL estimated from band intensities in SDS-PAGE was 2.4 (rhodanese) and 2.5
(MDH). Saturation of substrate protein binding sites of GroEL by this
procedure was confirmed as described later.
GroES Binding to the Free and Loaded GroELThe fluorescently
labeled GroESN increases its fluorescence upon binding to GroEL.
The extent of increase is
25%, regardless of nucleotides and the presence
or absence of unfolded proteins (Fig.
1, data not shown for ADPhex and AMPPNPhex).
Using the fluorescence increase of GroESN as a probe, we measured
time courses of binding of GroES to GroEL. GroESN bound rapidly to
unloaded GroEL in ATP, ADPhex, and AMPPNPhex with ATP
being the fastest and ADPhex the second fastest
(Fig. 2A). Binding
rates of GroESN in ADPraw and AMPPNPraw were
slightly faster than those treated with hexokinase
(Table I). The binding
reactions for all nucleotides are simulated by single exponential curves, and
the calculated association rate constants (kon) are in the
same order of magnitude, 107 M1
s1 (Table
I). The kon value of the GroESN
binding to unloaded GroEL in ATP (7.5 x 107
M1 s1) is consistent
with the previously reported values 4 x 107
M1 s1
(13) and 5 x
107 M1 s1
(4), ensuring the validity to
use GroESN for the binding experiments. These results show that ADP
and AMPPNP are as effective as ATP in mediating GroES binding to free GroEL.
However, these nucleotides have a very different effect on the
GroESN binding to the loaded GroEL
(Fig. 2, BD and
Table I). In ATP,
GroESN binding to the rhodanese-loaded GroEL and MDH-loaded GroEL
were slightly slower than that to free GroEL but still very rapid, completed
within 1 s (kon > 107
M1 s1)
(Fig. 2B). In
AMPPNPraw, the rates were slowed down by
102-fold
(Fig. 2C). In
AMPPNPhex, the rates were slowed down further; GroES binding to the
MDH-loaded GroEL and the rhodanese-loaded GroEL were reached only 40 and 5%
after 5 min, respectively. Similarly, the binding of GroESN to the
loaded GroEL was slow in ADPraw and even slower in
ADPhex (Fig.
2D). Although these slow GroES binding to the loaded
GroEL in ADP and AMPPNP have not been known previously, this can be expected
because GroES and unfolded protein compete for the overlapping binding sites
on the GroEL. Rather, a new mechanism is required to understand this rapid
GroES binding in ATP to the loaded GroEL.

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FIG. 1. Fluorescence change of GroESN induced by binding to
GroEL. Fluorescence spectra of GroESN with excitation
wavelength at 340 nm are shown. none, in the absence of GroEL;
none/ATPsingle, GroEL-GroESN formed in
ATPsingle; rho/ATPsingle,
rhodanese-loaded GroEL-GroES formed in ATPsingle. Spectra of
GroEL-GroESN formed in ADPhex and in
AMPPNPhex were also measured but not shown because they overlapped
with those of none/ATPsingle completely.
Experimental details are described under "Materials and Methods."
Em, emission; A.U., arbitrary units.
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FIG. 2. Binding of GroES to free GroEL and the loaded GroEL in various
nucleotides. Binding was monitored with the fluorescence change of
GroESN. The reactions were initiated by mixing the solution
containing GroESN and nucleotide with the solution containing free
GroEL or the loaded GroEL. Final concentrations of GroEL, GroESN,
and nucleotides were 0.1 µM, 0.02 µM, and 1
mM, respectively. Single exponential fitting curves that were used
to obtain association rate constants in
Table I are shown in white
lines. A, GroESN binding to free GroEL in ATP,
AMPPNPhex, and ADPhex. A.U., arbitrary units.
B, GroESN binding to the rhodanese-loaded GroEL and the
MDH-loaded GroEL in ATP. C, GroESN binding to the
rhodanese-loaded GroEL and the MDH-loaded GroEL in AMPPNPraw and
AMPPNPhex. D, GroESN binding to the
rhodanese-loaded GroEL and the MDH-loaded GroEL in ADPraw and
ADPhex.
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TABLE I Kinetic constants of GroES binding and release
The apparent association rate constants were obtained by fitting the curves
of GroESN binding to GroEL as typically shown in
Fig. 2. The values of
kon were calculated from linear fitting to the apparent
rate constants at several GroEL concentrations (see "Materials and
Methods"). Dissociation rate constants were obtained from the data in
Fig. 4.
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FIG. 4. Release of GroES from the GroEL-GroES complex. Release of GroES was
assessed by the loss of fluorescent GroESC from the
GroEL-GroESC complexes, which had been formed in
ATPsingle, AMPPNPhex, or ADPhex. Twenty-fold
molar excess of native GroES was added at time 0. Aliquots were loaded to gel
permeation HPLC at the indicated times, and the remaining GroESC in
the GroEL-GroES fraction was measured. Release of GroESC from the
GroEL-GroESC-rhodanese cis ternary complex formed in
ATPsingle is also shown. rho/ATPsingle,
rhodanese-loaded GroEL-GroES formed in ATPsingle.
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When the substrate binding sites of GroEL are not saturated with unfolded
proteins, it is predicted that rapid GroES binding in ADPhex to
free GroEL rings in partially loaded GroEL can be observed. Indeed, in
ADPhex, GroESN bound rapidly to two-thirds of the 1:1
(molar ratio of rhodanese and GroEL) rhodanese-loaded GroEL and to a quarter
of the 2:1 rhodanese-loaded GroEL (Fig.
3). The rapid binding was no longer observed for the 2.5:1
rhodanese-loaded GroEL, indicating that all the substrate binding sites of
GroEL were occupied by unfolded rhodanese. These results showed that 2.5
rhodanese and MDH can bind to GroEL even if GroEL has only two rings for
substrate protein binding. This discrepancy may be due to the error in
estimation of protein concentration or the occasional binding of two substrate
proteins to one GroEL ring as reported using citrate synthase as substrate
protein (14).

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FIG. 3. GroES binding to the partially loaded GroEL. Binding was monitored
with the fluorescence change of GroESN. Binding reactions were
initiated by mixing the solution containing GroESN and
ADPhex with the solution containing the rhodanese-GroEL complexes
with rhodanese:GroEL molar ratios 1:1, 2:1, and 2.5:1. Final concentrations of
GroEL, GroESN, and ADPhex were 0.05 µM,
0.05 µM, and 1 mM, respectively. A.U.,
arbitrary units.
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Dissociation of GroES from GroELDissociation of GroES from
GroEL was measured by the exchange of fluorescently labeled GroESC
bound on GroEL with an excess amount of unlabeled GroES. As shown in
Fig. 4, the
GroEL-GroESC complexes formed in ATPsingle and
ADPhex without substrate protein are surprisingly stable; only 10%
of the GroEL-GroESC complexes released GroESC after 1
week. The GroEL-GroESC complex formed in ATPsingle
becomes identical to that in ADPhex since the bound ATP in the
complex is hydrolyzed to ADP. This result shows the extraordinary stability of
the GroEL-GroESC complexes in ADP. The complex without substrate
protein formed in AMPPNPhex was relatively unstable and decayed in
2 days. The GroEL-GroESC-rhodanese complex formed in
ATPsingle was slightly less stable than the GroEL-GroESC
complex in ATPsingle in the absence of unfolded protein. This
instability in the presence of rhodanese may be due to the stimulation of
GroES release by substrate protein binding to trans ring
(4). Calculated dissociation
rate constants (koff)
(Table I) are smaller than the
previously reported value (3.8 x 105 s)
estimated from the dissociation of ADP moiety from the GroEL-GroES complex
(15). The reason of this
discrepancy is not known, but there is a possibility that a trace amount of
contaminated ATP would stimulate the exchange of bound ADP. The estimated
dissociation constants of GroES binding to GroEL are extremely small (
10
fM) and comparable with the dissociation constant of the biotin-avidin
binding. This strong GroES binding may enable GroES to bind the loaded GroEL,
the GroES binding site of which is covered by substrate protein.
cis Folding of RhodaneseFrom the slow binding of GroES to
the rhodanese-loaded GroEL in ADPhex and AMPPNPhex, it
can be predicted that formation of the cis ternary complex and the
cis folding cannot occur in these nucleotides. To examine this, we
compared the effect of these nucleotides on the chaperonin-assisted folding of
rhodanese. Since it has been known that rhodanese folds in the cis
cavity but does not fold spontaneously
(16), all the recovered
rhodanese activity can be attributed to the result of the cis
folding. The reaction was started by mixing the rhodanese-loaded GroEL, GroES,
and nucleotides (Fig.
5A). In ATP, the yield of recovered rhodanese reached to
more than 80% of the total bound rhodanese in 60 min. In ATPsingle,
the recovered rhodanese was nearly 50%, showing that unfolded rhodanese bound
in one GroEL ring is folded in the cis cavity after single ATP
hydrolytic cycle was terminated. We observed significant recovery of rhodanese
activity also in ADPraw (
50%) and AMPPNPraw
(
20%). The same results were reported previously by others
(79),
and it has been thought that ADP and AMPPNP can substitute ATP to some extent
for the chaperonin function. However, hexokinase treatment diminished
rhodanese activity in ADPhex and AMPPNPhex
(Fig. 5A). To
determine which is the cause of ATP contamination, contaminated ATP or ATP
production by contaminated adenylate kinase, rhodanese activity was measured
using pre-hexokinase-treated ADP and AMPPNP (ADPpre-hex and
AMPPNPpre-hex) that were treated with hexokinase and separated from
hexokinase by ultrafiltration (Fig.
5B). Approximately 50% of rhodanese was recovered in
ADPpre-hex. The initial lag period in the rhodanese recovery in
ADPpre-hex can be interpreted as the time required for the
accumulation of ATP by adenylate kinase. Indeed, in the presence of Ap5A, a
potent inhibitor of adenylate kinase
(17), rhodanese reactivation
disappeared in ADPraw, whereas it was not affected in ATP. The
reason for no rhodanese recovery in AMPPNPpre-hex may be explained
by the low concentration of contaminated ADP that is not enough for adenyate
kinase to produce ATP. These results lead to conclusion that the causes for
rhodanese recovery in ADPraw and AMPPNPraw are trace
amounts of contaminated adenylate kinase and contaminated ATP,
respectively.

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FIG. 5. Recovery of rhodanese activities assisted by GroEL and GroES in various
nucleotides. Recovery yields were expressed as mol of recovered enzyme per
mol of GroEL. A, recovery in ATP, ATPsingle,
AMPPNPraw, ADPraw, AMPPNPhex, and
ADPhex. B, recovery in AMPPNPpre-hex and
ADPpre-hex that had been pretreated with hexokinase and effect of 1
mM Ap5A, an inhibitor of adenylate kinase. Ap5A was also added to
the reaction mixture for ATP as a control. Note that hexokinase was included
in the reaction mixtures for ATPsingle (after 3 s),
AMPPNPhex, and ADPhex during the reaction period
(A) but was removed from the reaction mixtures of
AMPPNPpre-hex and ADPpre-hex prior to the initiation of
the reaction (B). No rhodanese recovery was observed in the absence
of nucleotide (data not shown).
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Formation of the cis Ternary ComplexWe assessed the
formation of the cis ternary complexes by using protease treatment;
the substrate proteins entrapped in the cis cavity are protected from
the protease digestion, whereas those bound to the trans ring are
readily digested as well as free unfolded proteins
(2,
16). The samples incubated for
120 min as in Fig. 5A
were ultrafiltrated to remove free GroES, digested by proteinase K, and
ultrafiltrated to remove proteinase K and digested polypeptide. Three kinds of
GroEL complexes, GroEL, GroEL-GroES, and the GroEL-GroES-rhodanese
cis ternary complex, should exist after this procedure. The relative
populations of GroEL, GroES, and rhodanese were estimated from the band
intensities in the CBB-stained SDS-PAGE
(Fig. 6A). The
rhodanese-loaded GroEL contained 2.4 mol rhodanese/mol of GroEL (lane
1), and all rhodanese molecules were digested by proteinase K (lane
2). In ATP, the folded rhodanese molecules had been removed by
ultrafiltration, and GroEL-GroES complexes remained (lane 3). In
AMPPNPraw, ADPraw, and ATPsingle, significant
amount of proteinase K-resistant rhodanese were observed (lanes
46). Molar ratios among GroEL, rhodanese, and GroES in these three
lanes were 1.0:0.81.4: 0.71.1, indicating that the
GroEL-GroES-rhodanese cis ternary complex was formed in these
nucleotides. The rhodanese molecules held in the cis cavity of these
complexes had finished folding because rhodanese activity of these complexes
is equivalent to the estimated amount of rhodanese from SDS-PAGE
(Fig. 6A, lower
panel). In contrast, in AMPPNPhex and ADPhex, no
rhodanese and a faint amount of GroES were observed (lanes 7 and
8), suggesting that GroES cannot bind to the rhodanese-loaded GroEL
or can form only unstable GroEL-GroES-rhodanese ternary complex. Slow GroES
binding to rhodanese-loaded GroEL in AMPPNPhex and
ADPhex (Fig. 2, C and
D) can be due to the formation of such unstable complex.
Next, we used MDH as a substrate protein
(Fig. 6B). Molar
ratios among GroEL, MDH, and GroES in AMPPNPraw, ADPraw,
and ATPsingle (lanes 46) were
1.0:0.81.1:1.01.1, indicating the stoichiometric formation of
the cis ternary complexes as well as rhodanese. The results of
AMPPNPhex (lane 7) and ADPhex (lane 8)
are different from those of rhodanese. In AMPPNPhex, smaller
amounts of MDH than ATPsingle were encapsulated in the cis
cavity, whereas rhodanese was not encapsulated. In ADPhex, no MDH
band was observed as rhodanese; however, GroES was bound to GroEL in contrast
to rhodanese. Probably, MDH was gradually released from GroEL, and then GroES
bound to these newly available binding sites. Folding of MDH in the
cis cavity was assessed by measuring MDH activity after the MDH dimer
was formed by releasing the MDH monomer from the cis cavity using
EDTA to open the GroES cap (Fig.
6B, lower panel). Recovered MDH activity of each
sample roughly corresponds to the amount of MDH in SDS-PAGE. ADPhex
did not support cis folding of MDH, but AMPPNPhex did so
in some extent. This ATP analogue seems to mimic the action of ATP in this
case by a yet unknown
mechanism.3

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FIG. 6. Formation of the cis ternary complex in various
nucleotides. Rhodanese (A) and MDH (B) were used as
substrate proteins. Lane 1, substrate protein-loaded GroEL without
proteinase K (ProK) treatment. Lane 2, substrate
protein-loaded GroEL treated with proteinase K. Lanes 38,
substrate protein-loaded GroEL and GroES were subjected to the following
procedures: a 2 h-incubation in the indicated nucleotides, ultrafiltration
(100-kDa cut), proteinase K treatment, ultrafiltration (100-kDa cut), and
SDS-PAGE analysis. Nucleotides included during a 2 h-incubation were: lane
3, ATP; lane 4, AMPPNPraw; lane 5,
ADPraw; lane 6, ATPsingle; lane 7,
AMPPNPhex; lane 8, ADPhex. Another aliquot of
the solution after the second ultrafiltration was treated with EDTA to detach
GroES, and the activities of rhodanese and MDH released from the cis
cavity were measured to estimate the cis folding yields that were
expressed as mol of recovered enzyme per mol of GroEL. (lower
panels). Experimental details are described under "Materials and
Methods."
|
|
ConclusionsThe previously reported ability of ADP, less
efficient than that of ATP in the formation of the cis ternary
complex, has made the current conception of the ATP-dependent chaperonin
function ambiguous. However, our results clearly show that the cis
ternary complex is not formed and that the cis folding does not occur
in ADP. Also, cis folding of substrate proteins in ADP has been
caused by the contaminated adenylate kinase because its inhibitor, diadenosine
pentaphosphate, suppressed the cis folding of rhodanese. It should be
noted that the direct measurement of adenylate kinase activity contaminated in
the purified GroEL is hard to detect due to the immediate hydrolysis of
produced ATP by GroEL. In contrast, AMPPNP appears to be able to mimic the
action of ATP to some extent; it supports cis folding for MDH but not
for rhodanese. This work sheds light on the key question on the chaperonin
function; how can ATP enable rapid binding of GroES to the substrate
protein-loaded GroEL whose binding sites for GroES are occupied by the
substrate protein?
 |
FOOTNOTES
|
---|
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
To whom correspondence should be addressed. Fax: 81-45-924-5277; E-mail:
myoshida{at}res.titech.ac.jp.
1 The abbreviations used are: AMPPNP,
5'-adenylyl-
,
-imidodiphosphate; AMPPNPhex,
AMPPNP that is always exposed to hexokinase and glucose; AMPPNPraw
and ADPraw, commercial AMPPNP and ADP that have not been exposed to
hexokinase; ADPhex, ADP that is always exposed to hexokinase and
glucose; ATPsingle, ATP exposed to hexokinase at 3 s after
initiation of the reaction, by which time only a single turnover of ATP
hydrolysis of GroEL can occur; MDH, malate dehydrogenase; GroESN,
GroES(T19C) labeled by 5(2-iodoacetylaminoethyl) aminonaphthalene-1-sulfonic
acid; GroESC, GroES(98C) labeled by 5(2-iodoacetylaminoethyl)
aminonaphthalene-1-sulfonic acid; Ap5A, diadenosine pentaphosphate; DTT,
dithiothreitol; HPLC, high pressure liquid chromatography. 
2 Concentrations of GroES and GroEL are all expressed as heptamer and
tetradecamer, respectively, in this article. 
3 In AMPPNPhex and ADPhex, MDH in the MDH-loaded GroEL
appeared to be gradually released from GroEL, and then GroES bound tightly to
the newly available binding sites, because recovery of some MDH activity was
observed even before the addition of EDTA (data not shown). In this case,
recovery occurred much more slowly in AMPPNPhex than in
ADPhex, indicating higher affinity of MDH to GroEL in
AMPPNPhex than in ADPhex, which is consistent with
slower GroESN binding to MDH-loaded GroEL in AMPPNPhex
than in ADPhex (Fig. 2,
C and D). The small cis folding of MDH
in AMPPNPhex detected in Fig.
6B may be related to this relatively strong affinity of
MDH to GroEL in AMPPNPhex and accidental entrapping of MDH in the
cis cavity, whereas GroES competes for its binding sites. 
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Drs. T. Hisabori, E. Muneyuki, and H. Taguchi for
valuable discussion, Drs. K. Kinosita, T. Kawashima, and T. Masaike for the
use of the stopped-flow apparatus, and S. Murayama, T. Inoue, and R. Suno for
experimental assistance.
 |
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