©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Reversal by GroES of the GroEL Preference from Hydrophobic Amino Acids toward Hydrophilic Amino Acids (*)

Axelle de Crouy-Chanel , Abdelhamid El Yaagoubi , Masamichi Kohiyama , Gilbert Richarme (§)

From the (1) Génètique et Biochimie, Institut Jacques Monod, Université Paris 7, 2 place Jussieu, 75005 Paris, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 -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.


INTRODUCTION

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 -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.


EXPERIMENTAL PROCEDURES

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.


RESULTS

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 Karound 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 Kis lowered (the Kfor 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.


DISCUSSION

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 -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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-144275098; Fax: 33-144273580.


ACKNOWLEDGEMENTS

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.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.