From the
The chaperones GroEL/hsp60 are present in all prokaryotes and in
mitochondria and chloroplasts of eukaryotic cells. They are involved in
protein folding, protein targeting to membranes, protein renaturation,
and control of protein-protein interactions. They interact with many
polypeptides in an ATP-dependent manner and possess a peptide-dependent
ATPase activity. GroEL/hsp60 cooperates with GroES/hsp10, and the
productive folding of proteins by GroEL generally requires GroES, which
appears to regulate the binding and release of substrate proteins by
GroEL. In a recent study, we have shown that GroEL interacts
preferentially with the side chains of hydrophobic amino acids (Ile,
Phe, Val, Leu, and Trp) and more weakly with several polar or charged
amino acids, including the strongest
The chaperonin GroEL (cpn60) and its helper protein GroES
(cpn10) assist in the cellular folding, assembly, and translocation of
other proteins and prevent incorrect associations within and between
polypeptide chains during protein folding
(1, 2, 3, 4, 5, 6, 7, 8, 9) .
GroEL interacts with non-native proteins, binding one or two molecules
in a central cavity inside the two stacked heptameric rings of the
60-kDa GroEL subunits
(10, 11) . It presumably interacts
with unfolded or loosely folded proteins through hydrophobic segments
exposed in the bound protein
(6, 12, 13, 14) and allows formation of the native tertiary structure
through ATP-dependent cycles of transient release of the bound protein
involving its peptide-dependent ATPase activity
(6, 15, 16) . GroES is a single heptameric ring
of 10-kDa subunits which forms a 1:1 complex with GroEL by binding to
one end of the GroEL cylinder
(11, 16, 17) . It
is necessary for full chaperonin function and for cell viability
(6, 7, 18, 19) . GroES is believed to
coordinate the release of the bound segments of the folding protein
through a coordinate regulation of the GroEL ATPase
(15, 16, 20) .
Whereas GroEL interacts
preferentially with the side chain of hydrophobic amino acids (Ile,
Val, Leu, Phe, and Trp) and displays a weaker interaction with more
polar amino acids, including the
In summary, GroES shifts the amino acid specificity of GroEL
from hydrophobic amino acids to hydrophilic ones. The specificity of
GroEL for hydrophobic amino acids correlates with the proposed
interaction of the chaperonin with the hydrophobic parts of unfolded
proteins
(22) ( GroEL-hydrophobic segments interaction)
and is reminiscent of the hydrophobic specificity of other chaperones
such as DnaK (cpn70), BiP (cpn70) or SecB
(23, 28, 29) . The hydrophobic grip of the
chaperonin on its substrate protein might be important in preventing
the aggregation of folding intermediates (reviewed in Ref. 30) and in
controlling the hydrophobic collapse of the bound protein
(1, 2, 3, 4, 5, 6) . The
GroES-induced decrease in the GroEL specificity for hydrophobic amino
acids is in accordance with the inhibition by GroES of the binding to
GroEL of the hydrophobic probe anilinonaphtalenesulfonate
(13) and of several substrate proteins (reviewed in Ref. 4). It
would allow the concerted release from GroEL of the hydrophobic
segments of the bound protein and their mutual interaction for a
folding trial event driven by the hydrophobic collapse of the released
segments (hydrophobic segments-hydrophobic segments interaction). The
GroES-induced increase in the GroEL affinity for hydrophilic amino
acids would allow an interaction of the chaperonin with the hydrophilic
parts of the folding protein ( GroEL-hydrophilic segments
interaction). During the first steps of folding, this hydrophilic
grip would help to destabilize the folding intermediates
(30, 31) and would allow GroEL to keep in contact with its substrate
protein as the chaperonin looses its hydrophobic specificity upon
interaction with GroES. In the last steps of folding, as the
hydrophobic parts of the folding protein are buried in the hydrophobic
core
(4, 30, 31) , the hydrophilic grip of the
chaperonin on the surface of the substrate protein would permit small
structural adjustments for the attainment of the final tertiary
structure. As the folding protein attains its less flexible and more
compact native form, its hydrophilic surface would have a decreased
interaction with GroEL, with a consequent release of the native
protein.
GroEL interacts mainly with amino acids involved in the
formation of regular secondary structures. This is in accordance with
the early appearance of secondary structures during protein folding
(30, 31) and with the hypothesis that tertiary
structure is formed by the assembly of secondary structure elements
(32) . Since GroES appears to go through ATP-dependent cycles ot
transient release from GroEL
(18) , GroEL might handle
We thank Dr. K. Nagata (Chest Disease Research
Institute, Kyoto, Japan) for his help during the purification of GroEL,
Dr. O. Fayet (Laboratoire de Microbiologie et Génètique
Moléculaire, CNRS, Toulouse, France) for the gift of the GroES
hyperproducing strain, Dr. A. Ducruix (laboratoire de Biologie
Structurale, CNRS, Gif/Yvette, France) for critical reading of the
manuscript, and Dr. J. Garwood for corrections in the English language.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-helix and
-sheet
formers (Glu, Gln, His, Thr, and Tyr). In this study, we show that
GroES reduces the specificity of GroEL for hydrophobic amino acids and
increases its specificity for hydrophilic ones. This shift by GroES of
the GroEL specificity from hydrophobic amino acids toward hydrophilic
ones might be of importance for its function in protein folding.
-helix and
-sheet formers
(Glu, Gln, His, Lys, Arg, Thr, and Tyr)
(21) , we show in this
study that GroES reduces the specificity of GroEL for hydrophobic amino
acids and increases its specificity for hydrophilic ones. This
modulation by GroES of the GroEL affinity for the amino acid side
chains of substrate proteins might have important implications in
protein folding.
Purification of GroEL and GroES
GroEL was
purified as described in
(10) from the hyperproducing strain of
Escherichia coli KY 1603 (R40-1) from the laboratory of
Dr. T. Yura (Institute for Virus Research, Kyoto University), and GroES
was purified from strain OFB 2806 from Dr. O. Fayet
(19) , as
described
(16) . Both proteins were dialyzed against 50
mM Tris-hydrochloride pH 7.4, 5 mM 2-mercaptoethenol.
ATPase Assay
1 µl of purified GroEL (1 pmol in
14-mer) in 50 mM Tris-hydrochloride, pH 7.4, 60 mM
KCl, 60 mM sodium phosphate, 5 mM 2-mercaptoethanol,
20% glycerol, was incubated for 1 h at 20 °C with 1 µl of 100
µM [H]ATP (7 Ci/mmol) containing 10
mM MgCl
and 1 µl of amino acid, in the absence
or in the presence of GroES at a final concentration of 2
µM in 7-mer. The reaction was linear as a function of
time, and it was terminated by applying 2 µl of sample to
polyethylene cellulose thin layer plates that had been spotted with
carrier nucleotide as described
(22) . Negligible amounts of AMP
were produced during the reaction, and GroES did not contain any
measurable ATPase activity even in the presence of amino acids.
Size Exclusion Chromatography
GroEL (4
µM in 14-mer), I-labeled R-CMLA (4
µM), GroES (30 µM in 7-mer), and amino acid
(5 mM) were loaded as described in 1.5 µl of 50
mM Tris, pH 7.4, 60 mM KCl, 60 mM sodium
phosphate, 3 mM MgCl
, 15% glycerol on a Bio-Rad
P-200 column (100-µl bed volume) equilibrated in 50 mM
Tris, pH 7.4, 60 mM KCl, 60 mM sodium phosphate, 3
mM MgCl
, 100 µg/ml serum albumin. 1-drop
fractions were collected (1 fraction/20 s) and counted for
radioactivity. Bovine R-CMLA was obtained from Sigma, and it was
labeled by the chloramine-T method as described
(23) .
Materials
ATP disodium salt and -casein were
from Sigma. [
H]ATP was obtained from Amersham
Corp. and was used at 1.5 Ci/mmol. L-Amino acids were used in
solutions adjusted to pH 7.4. All the other products were from Sigma
and were reagent grade.
Inhibition by GroES of the GroEL-Hydrophobic Amino
Acids Interaction
This inhibition is shown in Fig. 1. The
dependence of the GroEL ATPase on the concentration of Ile, Leu, and
Val was measured in the absence or in the presence of GroES. In the
absence of GroES, as previously reported, Ile, Leu, or Val stimulate
approximately 3-fold the GroEL ATPase with a Klower than 0.5 mM. In the presence of GroES, there is no
significant effect of Ile, Leu, or Val on the GroEL ATPase (the ATPase
activity of GroEL is inhibited by GroES, as reported by others (see
legend to Fig. 1)). Similarly, GroES decreases the specificity of
GroEL for Ala, Met, Phe, and Trp (see below). Thus, GroES may trigger a
conformational change in GroEL which decreases its interaction with the
hydrophobic amino acids, and which consequently would decrease its
interaction with the hydrophobic parts of substrate proteins.
Figure 1:
Interaction of GroEL and GroEL/GroES with
hydrophobic amino acids. The GroEL ATPase was measured as described
under ``Experimental Procedures'' at the concentrations of
Ile (,
), Leu (
,
), or Val (
,
)
indicated in the abscissa, in the absence ( open
symbols) or in the presence ( closed symbols) of GroES.
Each point represents the mean value of three experiments. A relative
activity of 1 represents the unstimulated activity of GroEL, which
amounts to 10 nmol ADP/min/mg of protein in the absence of GroES, and
6.5 nmol/min/mg of GroEL in the presence of
GroES.
Stimulation by GroES of the GroEL-Hydrophilic Amino Acids
Interaction
This stimulation is shown in Fig. 2. The
dependence of the GroEL ATPase on the concentration of the hydrophilic
amino acids Glu, Gln (-helix formers), and Thr (
-sheet
former) was measured in the absence or in the presence of GroES. In the
absence of GroES, as previously reported, Glu, Gln, or Thr stimulate
GroEL approximately 2-fold with a K
around 1 mM. In the presence of GroES, and in contrast
with what is observed with the hydrophobic amino acids, the interaction
of GroEL with Glu, Gln, and Thr is strengthened. The stimulation factor
is higher (approximately 3-fold) and (or) the K
is lowered (the K
for Gln shifts
from 1 mM to 0.4 mM). Thus, GroES may induce a
conformational change in GroEL, which improves its interaction with the
hydrophilic amino acids Glu, Gln, and Thr, and consequently could
strengthen its interaction with the hydrophilic parts of substrate
proteins.
Figure 2:
Interaction of GroEL and GroEL/GroES
with hydrophilic amino acids. The experiments were made as described
under ``Experimental Procedures'' at the final concentrations
of glutamate (,
), glutamine (
,
), or threonine
(
,
) indicated in the abscissa, in the absence
( open symbols) or in the presence ( closed symbols) of
GroES.
Interaction of Amino Acids with the Peptide-binding Site
of GroEL
To ascertain that the free amino acids exert their
effect by interacting with the peptide-binding site of GroEL, we
studied their ability to compete for protein binding to GroEL. Reduced
carboxyl-methylated -lactalbumin (R-CMLA), which maintains an
extended conformation in the absence of denaturant and possesses little
secondary structure, was chosen as the most versatile protein for this
study. Since this protein has been reported not to interact with GroEL
(7, 24) , we developed a rapid chromatographic procedure
(small columns with a bed volume of 100 µl and a 2 min retention
time for GroEL) to reduce the dissociation of protein-chaperone
complexes. As shown in Fig. 3 A, in these conditions,
R-CMLA (4 µM) appears to bind efficiently (O.6 mol/mol) to
GroEL (4 µM). In the presence of 5 mM isoleucine,
this binding is reduced to 40%, while it is not affected by 5
mM glutamine. The same competition experiments were conducted
in the presence of GroEL (4 µM) and GroES (30
µM) (Fig. 3 B). In the presence of GroES,
R-CMLA binding is weaker (30% of the binding measured in the presence
of GroEL alone). In contrast to what happens with GroEL alone, the
binding of R-CMLA to GroEL/ES is reduced to 10% in the presence of 5
mM glutamine, while it is not significantly affected by 5
mM isoleucine (Fig. 3 B). Competition
experiments with other amino acids (Fig. 3 C) indicate
that hydrophobic amino acids (Ile, Val, and Phe) compete for R-CMLA
binding to GroEL, while hydrophilic amino acids (Thr, Glu, and Gln)
compete for R-CMLA binding to GroEL/ES. Thus, in accordance with the
ATPase stimulation experiments presented above, GroEL seems to interact
preferentially with the hydrophobic amino acids of substrate proteins
and GroEL/ES with their hydrophilic amino acids.
Figure 3:
Amino
acid inhibition of R-CMLA binding to GroEL. A, GroEL (4
µM in 14-mer), I-labeled R-CMLA (4
µM), and amino acid (5 mM) were preincubated for
15 min at 20 °C and chromatographied on a Bio-Rad P-200 column.
Fractions were collected and counted for radioactivity. B,
same as A in the presence of GroES (30 µM in
7-mer). R-CMLA alone (
); chaperone(s) and R-CMLA (
);
chaperone(s), R-CMLA, and Ile (
); chaperone(s), R-CMLA, and Gln
(
). C, amount of R-CMLA bound to GroEL in the presence
of 5 mM amino acid, relative to the amount bound in the
absence of amino acid, in the absence ( spotted bars) or in the
presence ( black bars) of GroES.
Amino Acid Specificity of GroEL in the Absence and in the
Presence of GroES
The effects of GroES on the specificity of
GroEL for the 20 amino acid are summarized in Fig. 4(the amino
acids are ranked in decreasing order of hydrophobicity)
(25) and in Fig. 5(amino acids ranked as a function of
their secondary structure propensity)
(26, 27) . The
abscissae represent the stimulation of the GroEL ATPase by
each amino acid at a concentration of 0.7 mM. In the absence
of GroES (Fig. 4, spotted bars), and in accordance with
our previous results, the specificity of amino acids for GroEL
correlates with their hydrophobicity. The most hydrophobic amino acids
(at the bottom of the figure) give the strongest stimulation of the
GroEL ATPase. This hydrophobic interaction might be involved in the
interaction of the chaperone with the hydrophobic segments of folding
proteins. In the presence of GroES (Fig. 4, black bars),
the hydrophobic amino acids interact weakly (Met, Phe, and Trp) or not
at all (Ile, Leu, Val, and Ala) with GroEL, while several hydrophilic
amino acid interact more strongly with GroEL (Tyr, Thr, His, Glu, Gln,
Arg, and Lys). The decrease of the hydrophobic interactions between
GroEL and the hydrophobic segments of the bound protein could permit
these hydrophobic segments to interact with each other, thus allowing a
renaturation trial event of the folding protein. The increase in the
hydrophilic interactions of GroEL with the folding protein would allow
the chaperonin to have a hydrophilic grip on the accessible surface of
the partially folded protein. This would permit a recapture of its
structural elements for an additional denaturation/renaturation trial.
Figure 4:
Interaction of GroEL and GroEL/GroES with
the 20 amino acids (hydrophobicity ranking). The amino acids are ranked
(in a decreasing order of hydrophobicity from bottom to top) according
to the consensus hydrophobicity scale taken from Ref. 25. The GroEL
ATPase activity was measured as described under ``Experimental
Procedures'' in the presence of each amino acid at 0.7
mM, in the absence ( spotted bars) or in the presence
( black bars) of GroES. A relative activity of 1 represents the
unstimulated activity of GroEL, which amounts to 10 nmol/min/mg of
protein in the absence of GroES and 6.5 nmol/min/mg of GroEL in the
presence of GroES. The stimulation factors of the GroEL ATPase are the
mean values from three independent
experiments.
Figure 5:
Interaction of GroEL and GroEL/GroES with
the 20 amino acids (secondary structures propensity ranking). The
-helix formers are grouped together at the bottom of the figure,
followed by the
-sheet formers, and the
-turn formers at the
top of the figure. For the sake of clarity, the amino acids of each
group are subgrouped into hydrophobic and hydrophilic amino acids, and
the hydrophobic amino acids are boxed. The conformational
preference of amino acids are taken from Ref. 27. The GroEL ATPase was
measured as described under ``Experimental Procedures'' in
the absence ( spotted bars) or in the presence ( black
bars) of GroES.
When the amino acids are ranked according to their secondary
structure propensity
(26, 27) (Fig. 5), it
appears that GroES shifts the amino acid specificity of GroEL from the
hydrophobic -helix formers (Leu and Met) and
-sheet formers
(Ile, Val, Trp, and Phe) to the hydrophilic
-helix formers (Glu,
Gln, and His) and
-sheet formers (Thr and Tyr). It is worth noting
that Glu and Gln (hydrophilic
-helix formers) and Thr and Tyr
(hydrophilic
-sheet formers) are those amino acids whose
interaction with GroEL is stimulated in the presence of GroES. In
contrast, the
-turn formers Asp, Asn, and Ser do not appreciably
interact with GroEL, either in the absence or presence of GroES.
Interestingly, the
-turn forming amino acid proline, whose side
chain is chemically hydrophobic but whose location in native proteins
is that of a hydrophilic one, interacts more strongly with GroEL in the
presence of GroES (similar to the hydrophilic amino acids). This
interaction might be used by the chaperonin to handle the hydrophilic
proline turns which are exposed at the surface of the folding protein.
-helices or
-sheets, by their hydrophobic face, or the
hydrophilic face, in a cycle regulated by GroES, and help to adjust
them relative to each other for the attainment of the native tertiary
structure.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.