GroEL Recognizes an Amphipathic Helix and Binds to the Hydrophobic Side

GroEL is an essential Escherichia coli molecular chaperon that uses ATP to facilitate correct folding of a range of proteins in a cell. Central to the GroEL substrate diversity is how GroEL recognizes the substrates. The interaction between GroEL and substrate has been proposed to be largely hydrophobic because GroEL interacts with proteins in non-native conformations but not in native forms. Analysis of GroEL substrate proteins reveals that one of its main substrates are proteins with αβ folding domains, suggesting that GroEL may stabilize the collapsed αβ core by binding to hydrophobic surfaces that are usually buried between the α and β elements. In this study, we characterize the interaction between GroEL and a peptide derived from our previous selection via a phage display method. NMR studies map the peptide-binding site to the region containing Helices H and I, which is consistent with evidence that this region comprises the primary substrate-binding site. The peptide is largely unstructured in solution but adopts a helical conformation when bound to the GroEL apical domain with a moderate affinity (K_d = 17.1 ± 2.5 μm). The helical conformation aligns residues to form an amphipathic structure, and the hydrophobic side of this amphipathic helix interacts with GroEL as suggested by fluorescence quenching studies. Together with previous structural studies on the GroEL-peptide complexes, our work supports the notion that the amphipathic secondary elements in the substrate proteins may be the structural motif recognized by GroEL.The bacterial chaperonin GroEL and its co-chaperonin GroES are essential for cell viability by assisting folding of a wide range of proteins via an ATP-dependent mechanism (1–3). Structurally, fourteen 57-kDa GroEL subunits assemble into two back-to-back stacking heptameric rings, giving rise to two functionally independent central cavities (4). Each GroEL subunit folds into three distinctive domains: equatorial domain, intermediate domain, and apical domain. The equatorial domains contain the ATP-binding sites and provide most of the intra-ring interactions and all the inter-ring interactions. The apical domains form the rims of the central cavities and contain the binding sites for the substrate proteins and GroES. The intermediate domains link the apical domains and the equatorial domains. For the co-chaperonin GroES, seven GroES subunits, of 10 kDa each, assemble into a heptamer ring (5, 6). In forming the GroEL-GroES complex, GroES caps one end of GroEL, and large structural changes are observed in both GroEL and GroES (7). In GroEL, the apical domain is rotated 90° along its axis and 60° upwards, and the intermediate domain is closed down ∼25° to the equatorial domain. A loop in GroES (residues 17–33) that is unstructured in the isolated GroES adopts a β-turn structure and forms contact with the GroEL apical domain. Compared with the unliganded GroEL, the volume of the enclosed GroEL-GroES cavity is doubled, and the surface lining the wall of the GroEL cavity changes from hydrophobic to hydrophilic.A wealth of information derived from both intensive biochemical and structural characterizations has revealed a general role of GroEL-GroES in assisting protein folding (see reviews in Refs. 3, 8, and 9). Briefly, GroEL binds the substrate proteins in their aggregation-prone non-native states, preventing them from aggregating. Binding of ATP to the substrate occupied GroEL ring (cis-ring) presumably induces large conformational change in GroEL that promotes binding of GroES to the cis-ring. As a result of ATP and GroES binding, the substrate protein is displaced into the GroEL central cavity, initiating the folding process. Both hydrolysis of ATP in the cis-ring and binding of ATP to the substrate unoccupied ring (trans ring) weaken the GroES-GroEL interaction, and ATP binding to the trans ring results in the dissociation of GroES from GroEL, releasing substrate from the central cavity of GroEL. The released substrate may continue folding into the native state if in a folding competent state or may rebind to GroEL if it is still misfolded.One of the most intriguing aspects of the GroE-assisted folding is the substrate promiscuity. It has been shown that about 300 Escherichia coli proteins can interact with GroEL, and these proteins are diverse in terms of both structures and functions (10). A range of techniques have been applied to investigate this important yet complex aspect, and salient features regarding GroEL-substrate interactions have emerged. The apical domains, on the rim of the GroEL central cavity, contain the main substrate-binding site (11–13). Structural flexibility, reflected by both high temperature factors of the apical domain in the crystal structure of tetradecameric GroEL (14) and conformational multiplicity around Helix H and I (15), is proposed to account for the diverse spectrum of GroEL substrates. Mutational studies on GroEL suggest that the GroEL-substrate interactions are largely hydrophobic (16). Structural study on GroEL-substrate interaction, however, is hindered mainly because of the multiple conformations of the bound substrate protein. Very recently, NMR techniques have been used to directly investigate the bound conformations of the substrate (17, 18); yet the nature of GroEL-substrate interaction is not revealed. Peptides may mimic segments of substrate proteins, and studies of GroEL-peptide interactions have uncovered detailed intermolecular interactions and provided insights into principles of substrate recognition by GroEL. The bound peptides may adopt α-helix (19–23), β-hairpin (15), or extended conformations (24), and despite different conformations, they all appear to bind to Helix H and I of GroEL. Hydrophobic interaction dominates the interface between GroEL and peptides in either β-hairpin or extended structures and is proposed so between GroEL and α-helical peptides. These detailed structural characterizations on GroEL-peptide interactions have contributed to dissecting the complex nature of the substrate recognition by GroEL (25).We previously identified a high affinity peptide (strong binding peptide (SBP))² for GroEL using a phage display method and found that SBP adopts a β-hairpin structure bound to GroEL (15, 26). To investigate the contribution of the β-turn in SBP to the GroEL-SBP interaction, we have created various SBP variants with the intension to disrupt the β-turn structure and have studied their binding to GroEL. One of the peptides (termed SBP-W2DP6V), however, adopts a helical conformation when bound to GroEL by NMR analysis. NMR results also map the peptide-binding site on GroEL to be a region formed by Helix H and I. The helical peptide has an amphipathic feature, and fluorescence studies provide direct evidence that the hydrophobic face is involved in the interaction with GroEL. Our structural analysis, combined with previous studies, suggests that GroEL recognizes the amphipathic property in the secondary structures of the substrate protein and binds preferably to the hydrophobic side of these structural elements to stabilize and preserve their structures.