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GroEL Recognizes an Amphipathic Helix and Binds to the Hydrophobic Side.

ABSTRACT: 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 alphabeta folding domains, suggesting that GroEL may stabilize the collapsed alphabeta core by binding to hydrophobic surfaces that are usually buried between the alpha and beta 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 microm). 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.

SUBMITTER: Li Y

PROVIDER: S-EPMC2640968 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:One of the fundamental features of biomembranes is the ability to fuse or to separate. These processes called respectively membrane fusion and fission are central in the homeostasis of events such as those related to intracellular membrane traffic. Proteins that contain amphipathic helices (AHs) were suggested to mediate membrane fission via shallow insertion of these helices into the lipid bilayer. Here we analyze the AH-containing proteins that have been identified as essential for membrane fission and categorize them in few subfamilies, including small GTPases, Atg proteins, and proteins containing either the ENTH/ANTH- or the BAR-domain. AH-containing fission-inducing proteins may require cofactors such as additional proteins (e.g., lipid-modifying enzymes), or lipids (e.g., phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidic acid [PA], or cardiolipin). Both PA and cardiolipin possess a cone shape and a negative charge (-2) that favor the recruitment of the AHs of fission-inducing proteins. Instead, PtdIns(4,5)P2 is characterized by an high negative charge able to recruit basic residues of the AHs of fission-inducing proteins. Here we propose that the AHs of fission-inducing proteins contain sequence motifs that bind lipid cofactors; accordingly (K/R/H)(K/R/H)xx(K/R/H) is a PtdIns(4,5)P2-binding motif, (K/R)x6(F/Y) is a cardiolipin-binding motif, whereas KxK is a PA-binding motif. Following our analysis, we show that the AHs of many fission-inducing proteins possess five properties: (a) at least three basic residues on the hydrophilic side, (b) ability to oligomerize, (c) optimal (shallow) depth of insertion into the membrane, (d) positive cooperativity in membrane curvature generation, and (e) specific interaction with one of the lipids mentioned above. These lipid cofactors favor correct conformation, oligomeric state and optimal insertion depth. The most abundant lipid in a given organelle possessing high negative charge (more negative than -1) is usually the lipid cofactor in the fission event. Interestingly, naturally occurring mutations have been reported in AH-containing fission-inducing proteins and related to diseases such as centronuclear myopathy (amphiphysin 2), Charcot-Marie-Tooth disease (GDAP1), Parkinson's disease (?-synuclein). These findings add to the interest of the membrane fission process whose complete understanding will be instrumental for the elucidation of the pathogenesis of diseases involving mutations in the protein AHs.

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GroEL Recognizes an Amphipathic Helix and Binds to the Hydrophobic Side.

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