Amino Acid Specificity of the Escherichia coli Chaperone GroEL (Heat Shock Protein 60)"

The chaperones GroEUhspBO are present in all pro- karyotes and in mitochondria and chloroplasts of eu-karyotic cells. They are involved in protein foiding, pro- tein 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. The nature of the structural elements of substrate proteins recognized by GroEUhsp60 is still unknown. In this study, we show that the GroEL chaperone of Escherichia coli interacts with single amino acids. The hydrophobic amino acids ne, Phe, Val, Leu, and "qJ present the strongest interaction with GroEL. While most of these hydrophobic amino acids are p-sheet formers, GroEL interacts also with the a-helix formers Glu, Ala, Gln, and His. The multiple interactions of GroEL with the side chains of hydrophobic and polar amino acids, including the strongest a-helix and P-sheet formers would allow this chaperone to act as an amphiphilic organizer of protein folding.

Chaperones constitute a class of polypeptide-binding proteins which are involved in protein folding, protein targeting to membranes, protein renaturation, and control of protein-protein interactions (reviewed in Refs. 1-4). The two main classes of chaperones (hsp70/DnaK and hspGO/GroEL) bind to segments of completely or partially unfolded polypeptides (4) and possess a peptide-dependent ATPase activity (5,G). The mechanisms of the binding of substrate proteins to chaperones are not precisely known. It has been suggested that chaperones might recognize unfolded polypeptide chains (71, molten globule conformation (8), secondary structures (7, 9), or hydrophobic sequences (6). In this report, we show that GroEL'hspGO interacts with single amino acids, and we suggest that amino acid side chains of substrate proteins would represent a simple and important structural element recognized by chaperones. In contrast to DnaK (which interacts with hydrophobic amino acids,l GroEL interacts not only with hydrophobic amino acids (Ile, Phe, Leu, Val, and "p), but also with less hydrophobic and with charged amino acids (Ala, %, Thr, Glu, Gln, His, Lys, Arg, and Pro). The common interaction of DnaK and GroEL with hydrophobic amino acids would allow their interaction with hydrophobic residues which are exposed in unfolded or partially denatured proteins (8,10,11). The interaction of GroEL'hspGO with hydrophobic and hydrophilic amino acids * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ATPase Assay-1 pl of purified GroEL (0.12 pmol) in 50 m~ Trishydrochloride, pH 7.4, 50 m~ KCl, 0.06 M sodium phosphate, and 5 m~ 2-mercaptoethanol was incubated for 1 h at 20 "C with 1 pl of 100 p~ r3H]ATP (1.5 Cilmmol) containing 300 p~ MgClz and 1 pl of amino acid as indicated. The reaction was linear as a function of time, and it was terminated by applying 2 pl of sample to polyethyleneimine cellulose thin layer chromatography plates that had been spotted with camer nucleotide as described (6). Negligible amounts of AMP were produced during the reaction. A relative activity of 1 represents 6 nmoYmidmg of protein.
Materials-ATP disodium salt and a-casein were from Sigma.
[3H]ATP was obtained from Amersham 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
Amino Acid Specificity of GroEL-The dependence of the GroEL ATPase on the concentration of three amino acids (Ile, Glu, and Gly) taken as an example, is shown in Fig. L4. The hydrophobic amino acid isoleucine stimulates GroEL %fold (K, = 0.2 m). The polar amino acid glutamate stimulates GroEL 3-fold (& = 0.7 mM) while glycine does not affect the ATPase activity of GroEL. a-Casein (a protein which possesses certain properties of partially denatured proteins) stimulates the GroELATPase %fold with a K, of 0.5 p~ (not shown). Thus, the stimulation factor of the GroEL ATPase by amino acids is similar to the stimulation factor of GroEL by a-casein or by other peptides (81, and the K, of the amino acid stimulation i s approximately 500-fold higher than the K, of the stimulation by a-casein (M, = 120,000), a protein composed of several hundreds of amino acids. Furthermore, the stimulation of the GroEL ATPase by Ile or Glu is not observed when GroEL is already stimulated by a-casein (Fig. 1B), suggesting that the stimulation of GroEL by amino acids involves the peptide-binding sites of GroEL.
The effects of the 20 amino acids on the ATPase activity of GroEL are summarized in Table I peptides could consist of a single (or a very few) amino acid side chain. The preferential interaction of chaperones with nonnative states of proteins would result from the exposure of a specific subset of amino acids (mainly hydrophobic amino acids) by non-native proteins, or from a greater accessibility of the amino acid side chains in unfolded proteins.
Interaction of GroEL with Arginine and Lysine-Since arginine and lysine produce an inhibition of the GroEL ATPase, in contrast to the other amino acids, their effects on GroEL were further investigated. As shown in Fig. 2, when assayed in the presence of a stimulating amino acid (tyrosine or isoleucine), lysine produces a stimulation of the GroEL ATPase instead of inhibiting it. Similar results were obtained with arginine (not shown). Thus, the ability of arginine and lysine to stimulate GroEL in the presence of other amino acids suggests that they are also implicated in the interaction of GroEL with peptides. Znteraction of GroEL with Amino Acid Derivatives-The interaction of GroEL with amino acid derivatives having their a-amino group blocked by acetylation, andor their a-carboxylic group blocked by esterification was investigated. As shown in Fig. 3, N-acetyl-L-tyrosine ethyl ester (with amino and carboxylic groups blocked) stimulates the GroEL ATPase in a similar manner as tyrosine (the stimulation factor is similar and the K, is slightly lower). This suggests that GroEL interacts with the side chain of amino acids, as was expected from the different specificities of GroEL for the 20 amino acids (which differ by their side chains). The decreased interaction of N-acetyl-L-ty-

Stimulation of GroEL by the 20 amino acids
The GroEL ATPase activity was measured as described in Fig. 1. A relative activity of 1 represents the unstimulated activity of GroEL, which amounts to 6 nmoVmidmg of protein. The stimulation (or inhibition) factors of the GroEL ATPase and the K, (or K,) are the mean values from three independent experiments. The standard deviations of the ATPase activities and of the K, (or K,) were, respectively, less than 15 and 25%. Data analysis was made by non-linear regression as described (25  rosine and tyrosine ethyl ester with GroEL is probably due to the net negative or positive charge carried by these amino acid derivatives. Similar results were obtained with leucine and tryptophan derivatives (not shown).
Amino Acid Hydrophobicity and GroEL Stimulation-The ability of amino acids to stimulate the GroELATPase correlates well with their hydrophobicity (Fig. 4). The most hydrophobic amino acids Ile, Phe, Val, Leu, and "rp give the strongest stimulation of the GroEL ATPase. As previously discussed for DnaWhsp70, the specific interaction of chaperones with hydrophobic amino acids (which are buried in native globular proteins and exposed in non-native forms (11)) would allow an interaction of these chaperones with nascent polypeptides or with denatured proteins (8,101. Such a hydrophobic interaction might also be involved in the interaction of chaperones with the hydrophobic sequences of nascent membrane proteins or with  Table I. signal sequences of exported proteins. The interaction of chaperones with hydrophobic amino acids of substrate proteins might involve either consecutive amino acids (signal sequences, hydrophobic sequences of membrane proteins) or non-consecutive amino acids (favoring the formation of collapsed folding intermediates), thus allowing a great flexibility in their interactions with substrate proteins.
As shown in Fig. 4 and in Table I, GroEL interacts also (although with lower affinity) with less hydrophobic, and with polar or charged amino acids. In particular, Thr, Pro, His, Glu, Implication of Amino Acids in Protein Secondary Structures and GroEL Stimulation-In Fig. 5, the amino acids have been ranked according to their ability to form secondary structures in proteins (15,16). The a-helix formers interact with GroEL and comprise both hydrophobic amino acids (Leu and Met) and less hydrophobic or polar and charged amino acids (Ala, Glu, Gln, His, Lys, and Arg). Glu and Gln stimulate GroEL despite their lack of hydrophobicity, and in contrast to Asp and Asn, which are involved in the formation of p-turns.
The a-sheet formers (Val, Ile, Tyr, Trp, Phe, and Thr) strongly stimulate GroEL. However, this result is confusing since the strongest p-sheet formers are also hydrophobic amino acids (17). Meanwhile, Tyr and Thr which are moderately hydrophobic interact with GroEL, while being important for the formation of amphiphilic p-sheets. The p-turn forming amino acids do not interact significantly with GroEL (Gly, Asn, Ser, and Asp) except proline which constitutes a unique case among amino acids in the formation of protein structure, and possesses the strongest a-helix and p-sheet breaking properties of all amino acids. Thus, GroEL appears to interact with amino acids that are frequently found in a-helices and 8-sheets.

DISCUSSION
Our results suggest that amino acid side chains of substrate proteins would be an important motif recognized by chaperones. While GroEL appears to interact with 15 amino acids, the Amino Acid Specificity of GroEL Chaperone number of different amino acid-binding sites is probably less, since similar amino acids are likely to interact with the same binding site (as in the case of proteases). Even if the number of amino acid-binding sites is reduced to about five on each GroEL protomer, this would make 70 amino acid-binding sites on each GroEL 14-mer, and the same number of possible interactions between the chaperone and its substrate protein, since each 14-mer appears to bind only one molecule of protein (8).
The ability of groEL to recognize hydrophobic residues, which are buried inside native proteins (11,18), and exposed in their non-native forms would allow the specific interaction of the chaperone with non-native proteins (unfolded states or molten globular states (19)) during protein folding, protein targeting to membranes, or protein denaturation (20-22). The hydrophobic interaction of GroEL and DnaK with unfolded proteins might be implicated also in the transfer of unfolded polypeptides from DnaK/hsp70 to GroEL/hspGO (12).
The interaction of GroEL with both hydrophobic and hydrophilic amino acids might be important for protein folding. GroEL could act as an amphiphilic organizer of protein folding, with its hydrophobic sites possibly located in the center of its double stacked rings of heptamers (4) and its hydrophilic sites in the peripheral parts. Multivalent reversible interactions of the substrate protein with the hydrophobic and hydrophilic sites (hydrophobic inside) would control the hydrophobic collapse and allow the substrate protein to experience many conformations (with the help ot the GroEL ATPase activity) before the attainment of its final conformation. It has been suggested that clustering of amino acid side chains might be important for pulling together secondary structures in the folding process (24); it is noteworthy that the highly preferred cluster residues (Trp, His, Arg, Tyr, Glu, Gln, and Phe (24)) strongly interact with GroEL. The folded protein might present a decreased interaction with the hydrophobic sites of GroEL resulting in a displacement of the protein to the surface of the chaperone.