(Received for publication, July 7, 1995; and in revised form, September 11, 1995)
From the
This study addresses the role of ATP-bound and free
Mg and Mn
ions in the activation
and modulation of chaperonin-assisted refolding of urea-denatured
malate dehydrogenase. As compared with Mg
,
Mn
ions caused a significant increase in the rate of
GroE-assisted malate dehydrogenase refolding and, concomitantly, a
decrease in the rate of ATP hydrolysis. Moreover, Mn
increases the affinity of GroES for GroEL, even in the presence
of saturating amounts of Mg
. Chemical cross-linking
showed that lower concentrations of Mn-ATP as compared with Mg-ATP are
needed to form both asymmetric GroEL
GroES
and
symmetric GroEL
(GroES
)
particles.
The manganese-dependent increase in the rate of protein folding
concurred with a specific increase in the amount of symmetric
GroEL
(GroES
)
particles detected in
a chaperonin solution. Thus, Mn
is a cofactor that
can markedly increase the efficiency of the chaperonin reaction in
vitro. Mn
ions can serve as an important tool
for analyzing the molecular mechanism and the structure of chaperonins.
In Escherichia coli, chaperonins GroEL and GroES assist
the folding and assembly of a large array of proteins (Goloubinoff et al., 1989a; Viitanen et al., 1992; Martin et
al., 1992; Horwich et al., 1993). Purified GroEL and GroES
oligomers assist the correct refolding of
nonnative polypeptides by preventing protein aggregation (Goloubinoff et al., 1989b; Buchner et al., 1991). Nonnative
proteins are proposed to interact with hydrophobic surfaces on the
GroEL
core chaperonin (Pelham, 1986; Goloubinoff et
al., 1989b, 1991; Fenton et al., 1994). Under
``nonpermissive'' conditions (Schmidt et al., 1994),
co-chaperonin GroES
is required for the release of the
bound protein from the chaperonin in a state that is committed to the
correct folding pathway (Goloubinoff et al., 1989b; Martin et al., 1991; Mendoza et al., 1991).
A full
understanding of the molecular mechanism by which GroEL and GroES
chaperonins assist the refolding of a nonnative protein requires
further analysis of the correlations between the affinity of ATP, ADP,
GroES, and unfolded proteins for the GroEL
and
GroEL
GroES
oligomers during the protein
folding reaction. In addition, the energy requirement of the protein
folding reaction needs further investigation. Under nonpermissive
conditions, protein folding absolutely depends on ATP hydrolysis
(Goloubinoff et al., 1989b; Schmidt et al., 1994).
Refolding of one protomer of rhodanese or Rubisco was estimated to
require the hydrolysis of approximately 130-100 ATP molecules
(Martin et al., 1991; Azem et al., 1995). However,
ATP hydrolysis does not reciprocally depend on protein folding, because
GroEL
alone or in association with GroES
hydrolyzes ATP even in the absence of unfolded proteins (Hendrix,
1979). An increase in the rate of ATP hydrolysis by GroEL
has been observed in the presence of guanidium HCl denatured
proteins (Martin et al., 1991). However, this increase has
been attributed to the guanidium HCl rather than to the unfolded
proteins (Todd and Lorimer, 1995). The protein folding process was
therefore suggested to take advantage of, rather than activate, a
futile cycle of ATPase by the chaperonin (Todd et al., 1993,
1994; Martin et al., 1993). Nevertheless, five GroEL mutants
with reduced ATPase but unaffected protein folding activities have been
reported (Fenton et al., 1994), pointing out that the energy
cost of the chaperonin folding reaction can be lowered. Therefore, the
possibility remains that the in vitro chaperonin reaction is
not optimal and can be improved by mutagenesis or by cofactors.
In
this study, we describe such a cofactor, Mn ions.
Even in the presence of excess Mg
, Mn
ions decrease the rates of ATP hydrolysis while at the same time
increasing the rate of protein folding and the affinity of GroES for
GroEL. Thus, Mn
significantly increases the
efficiency of the chaperonin reaction.
Figure 1:
Effect of Mn and
GroES on the GroEL ATPase. A, time-dependent hydrolysis of
ATP. GroEL (3 µM protomers) was incubated at 25 °C
with (circles) or without (squares) GroES (9
µM protomers) in the presence of 0.2 mM [
-
P]-ATP, 20 mM KCl, and 4
mM MgAc
(
and
) or MnAc
(
and
). The initial rates of the ATPase activity,
expressed as the number of turnovers per GroEL monomer, were derived
from a linear regression analysis. B, GroES-dependent
hydrolysis of ATP. GroEL (2 µM) was incubated for 17 min
at 37 °C with increasing concentrations of GroES in the presence of
1 mM ATP and 10 mM MgAc
(
) or 2
mM MnAc
with (
) or without (
) 10
mM MgAc
.
In the presence of saturating concentrations of ATP (1
mM) and Mg (10 mM), increasing
concentrations of GroES caused a maximal inhibition of 60% of the
GroEL-ATPase activity (Fig. 1B). Remarkably, the
addition of 2 mM Mn
further increased the
inhibition of the ATPase by GroES to 82%, approaching the level of
inhibition in the presence of 2 mM Mn
alone
(85%). Thus, Mn
acts as an inhibitor of the
magnesium-dependent ATPase activity of GroEL in the presence of GroES.
Figure 2:
Effect of Mn on
chaperonin-assisted protein folding. A, time-dependent protein
folding. Urea-denatured mMDH was diluted 70-fold to a final monomer
concentration of 257 nM in buffer containing GroEL (2
µM protomers), GroES (6 µM protomers), 2
mM MgAc
(
) or MnAc
(
), and
ATP (22.5 µM). Aliquots were assayed for mMDH activity at
the indicated time points. B, protein refolding depends on
Mg
or Mn
concentrations. Denatured
mMDH was diluted as in A in buffer containing GroEL (2
µM), GroES (3 µM), ATP (0.75 mM),
and increasing concentrations of MgAc
(
) or MnAc
(
). Samples were incubated for 8 min and assayed for mMDH
activity. C, ATP concentration-dependent refolding. Denatured
mMDH was diluted as in A in a buffer containing GroEL (2
µM), GroES (6 µM), MgAc
(2
mM) (
) or MnAc
(
), and increasing
concentrations of ATP. Samples were incubated for 8 min and assayed for
mMDH activity.
As in the case of the
GroEL ATPase, divalent ions are essential to the protein folding
activity of GroE chaperonins. In contrast to the 2-fold inhibition of
the chaperonin ATPase by Mn (Fig. 1), the
manganese-dependent rate of mMDH folding was twice as high as the
magnesium-dependent rate of protein folding (Fig. 2B).
Moreover, Mg
and Mn
activated mMDH
refolding in a biphasic manner. This suggests that chaperonin-dependent
protein folding is activated by micromolar amounts of either Mg-ATP or
Mn-ATP and also by millimolar amounts of free Mg
or
Mn
, as in the case of the GroEL
ATPase
(Diamant et al., 1995).
Figure 3:
ATP concentration dependence of GroES
binding to GroEL. GroEL (3 µM) was first incubated as in Fig. 2C with GroES (9 µM) in the presence
of 0, 2, 6, 25, and 75 µM ATP (lanes 1-5,
respectively) and MgAc (A) or MnAc
(B) and then cross-linked with glutaraldehyde and
separated on SDS-polyacrylamide gels (see ``Materials and
Methods'').
Increased affinity of GroES for GroEL in the
presence of Mn as compared with Mg
was demonstrated in Fig. 4. In the presence of excess
Mg
(20 mM), higher concentrations of GroES
were required to drive the chaperonin-dependent refolding of mMDH than
in the presence of the same amount of Mg
,
supplemented with 10-fold less Mn
(2 mM).
Thus, in the presence of Mg
alone, a GroES/GroEL
molar ratio (R
) of 1.1 could drive half of the
maximal rate of mMDH refolding, whereas in the presence of
Mg
and Mn
, a R
of only 0.75 was able to drive the same effect (Fig. 4A).
Figure 4:
Mn ions increase the
affinity of GroES
to GroEL
. Urea-denatured
mMDH was diluted as in Fig. 2in buffer containing increasing
amounts of GroES, GroEL (1.75 µM), ATP (0.75 mM),
and MgAc
(20 mM) or MgAc
(20
mM) supplemented by MnAc
(2 mM). A, GroES-dependent protein refolding activity and chaperonin
ATPase activity (inset) in the presence of MgAc
and MnAc
(
) or in the presence of MgAc
alone (
). B, GroES-dependent energy cost of
chaperonin reaction expressed as the number of ATP molecules hydrolyzed
per refolded mMDH in the presence of MgAc
and MnAc
(
) or in the presence of only MgAc
(
). Inset, GroES-dependent relative efficiency of chaperonin
activity in the presence of MgAc
and MnAc
compared with the efficiency in the presence of MgAc
alone (
). C, GroES-dependent formation of
GroEL
(GroES
)
chaperonin
hetero-oligomers in the presence of MgAc
and MnAc
(
) or in the presence of only MgAc
(
)
measured by cross-linking with glutaraldehyde and SDS electrophoresis
as in Fig. 3(gel not shown).
Under the same conditions, the effect of
Mn on the chaperonin ATPase activity in the presence
of increasing amounts of GroES was measured (Fig. 4B, inset). The number of ATP molecules hydrolyzed per refolded
mMDH (Fig. 4B) was found to be about 20 times higher
than in the case of rhodanese (Martin et al., 1991) or Rubisco
(Azem et al., 1995).
In addition, regardless of the nature
of the divalent ions, an increase in GroES concentration caused a
decrease of the energy cost of the folding reaction. Thus, in the
presence of Mg alone, the reaction was 74-fold less
efficient at R
= 0.4 than at R
= 2.0. Remarkably,
Mn
, even in the presence of a 10-fold excess of
Mg
, increased the efficiency of the reaction,
particularity when GroES was substoichiometric to GroEL. For example,
at R
= 0.4, the efficiency of the
reaction was 21-fold higher in the presence of 2 mM Mn
and 20 mM Mg
than
in the presence of 20 mM Mg
alone (Fig. 4B, inset). However, Mn
improved the chaperonin efficiency by 3-fold for all R
1.0.
Since the first in vitro assay for the
chaperonin-assisted refolding of a nonnative protein, Rubisco, by
GroEL, GroES
, and Mg-ATP (Goloubinoff et
al., 1989b), various additional co-factors have been described.
Thus, K
or ammonium ions are essential for ATP
hydrolysis and GroE-assisted protein folding (Viitanen et al.,
1990; Todd et al., 1993). ATP analogues inhibit protein
folding in the presence of chaperonins (Staniforth et al.,
1994; Miller et al., 1993). Here, we present evidence that
ATP-bound and free Mg
and Mn
ions
affect the affinity of GroES for GroEL and the rates of chaperonin
ATPase and of protein folding.
Micromolar amounts of ATP-bound
Mg or Mn
have been previously shown
to activate ATP hydrolysis by GroEL
(Azem et al.,
1994a; Diamant et al., 1995). In addition, millimolar amounts
of free Mg
and Mn
were shown to
stabilize the quaternary structure of the GroEL
(Azem et al., 1994a) and further activate the ATPase of
GroEL
(Diamant et al., 1995). Free Mn
was also suggested to interact with a high affinity allosteric
site on GroEL
, inhibiting the ATPase activity by half,
even in the presence of a large excess of Mg
ions
(Diamant et al., 1995).
We show here that Mn ions and GroES
use distinct mechanisms to inhibit the
ATPase activity of the GroEL
core oligomer. Despite this
inhibition, the rate of chaperonin-dependent refolding of mMDH was
higher in the presence of Mn
and an excess of
Mg
than in the presence of Mg
ions
alone. This implies that the efficiency of the chaperonin reaction can
be dramatically improved in vitro by a co-factor, such as
Mn
.
GroES directly controls the energy cost of the
chaperonin reaction. Thus, at R = 0.5,
the refolding of a mMDH molecule required the hydrolysis of 20 times
more ATP molecules than at R
= 1.25 (Fig. 4B). This difference is reduced to 2.9-fold when
2 mM Mn
is added to 20 mM Mg
. Chemical cross-linking revealed that at R
= 0.5, the chaperonin solution was
populated by a majority of asymmetric GroEL
GroES
particles (Azem et al., 1994b, 1995). In contrast, at R
= 1.25, the chaperonin solution was
populated by a majority of symmetric
GroEL
(GroES
)
particles (Fig. 4C).
Although Mn ions
increase the affinity of GroES
for both GroEL
and GroEL
GroES
particles, we found that
the rate of mMDH refolding precisely correlates with the amount of
symmetric GroEL
(GroES
)
particles
in the solution, confirming that the formation of symmetric
GroEL
(GroES
)
particles is
rate-determining for the protein folding reaction (Azem et
al., 1995). Consistent with the observation that Mn
increases the affinity of GroES
for the
GroEL
GroES
particle, protein folding was
dramatically improved, especially under substoichiometric amounts of
GroES
, when the symmetric particle was limiting. Thus,
Mn
improves the efficiency of protein folding by two
means: increasing the formation of symmetric
GroEL
(GroES
)
particles and
reducing the rates of ATP hydrolysis.
Both the protein substrate and
GroES have been suggested to go through mechanistically coupled cycles
of binding/release until the folding protein has lost its affinity for
GroEL (Martin et al., 1993). Furthermore, the binding/release
cycle of GroES has been shown to be coupled with the ATPase cycle (Todd et al., 1994). We found that Mn ions
decreased the rate of ATP hydrolysis but increased the affinity of
GroES for GroEL. From such a behavior, Mn
should
inhibit the ATP-dependent cycle of GroES binding/release on GroEL.
However, we found that Mn
increased the rates of
protein folding. We conclude that conditions may exist in vitro were the two cycles of protein and GroES binding/release are not
necessarily coupled mechanistically. We are now investigating the
effect of Mn
on the rates of GroES exchange on GroEL
during protein folding.