Basis of Substrate Binding by the Chaperonin GroEL

Abstract

The molecular chaperonins are essential proteins involved in protein folding, complex assembly, and polypeptide translocation. While there is abundant structural information about the machinery and the mechanistic details of its action are well studied, it is yet unresolved how chaperonins recognize a large number of structurally unrelated polypeptides in their unfolded or partially folded forms. To determine the nature of chaperonin-substrate recognition, we have characterized by NMR methods the interactions of GroEL with synthetic peptides that mimic segments of unfolded proteins. In previous work, we found using transferred nuclear Overhauser effect (trNOE) analysis that two 13 amino acid peptides bound GroEL in an amphipathic alpha-helical conformation. By extending the study to a variety of peptides with differing sequence motifs, we have observed that peptides can adopt conformations other than alpha-helix when bound to GroEL. Furthermore, peptides of the same composition exhibited significantly different affinities for GroEL as manifested by the magnitude of trNOEs. Binding to GroEL correlates well with the ability of the peptide to cluster hydrophobic residues on one face of the peptide, as determined by the retention time on reversed-phase (RP) HPLC. We conclude that the molecular basis of GroEL-substrate recognition is the presentation of a hydrophobic surface by an incompletely folded polypeptide and that many backbone conformations can be accommodated.

Perturbed ATPase activity and not “close confinement” of substrate in the cis cavity affects rates of folding by tail-multiplied GroEL

“…6 and 7). For substrates that are more tightly bound, however, presumably via a greater amount of exposed hydrophobic surface (8), a subsequent step of ATP/GroES binding to the substrate-bound ring ejects the substrate off the cavity wall into a now GroES-encapsulated and hydrophilic so-called cis cavity, where folding commences (9)(10)(11)(12). The reaction proceeds for Ϸ10 seconds before ATP hydrolysis in the cis ring followed by trans ring ATP binding ejects the cis ligands, including substrate polypeptide whether folded or not (11,13,14).…”

First glimpses of a chaperonin-bound folding intermediate

“…Results from peptide studies using transferred nuclear Overhauser effects (15)(16)(17) or crystallography (18) have established that various conformations can bind to GroEL; the common feature that mediates binding is exposure of a patch of hydrophobic surface (complementary to the binding surfaces on the apical domains of GroEL). Atomic structures (18,19) implicated two helices on the inner binding surfaces of the apical domains in polypeptide binding, and mutagenesis results (20) also suggest that hydrophobic residues on an underlying extended segment are crucial for substrate binding.…”

Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity

“…Additionally, the GroEL apical domain residues implicated in substrate binding are largely hydrophobic (14). Finally, previous studies on the basis of substrate interaction with GroEL using short model peptides have concluded that the most important determinant of substrate binding is the presentation of a cluster of hydrophobic residues (15)(16)(17).…”

“…The only evidence addressing the chiral specificity of GroEL/ ES comes from a study that qualitatively demonstrated binding of a short D-peptide to GroEL (16). However, this NMR study required peptide concentrations that greatly exceed physiologic levels and did not localize the interaction to the substratebinding region of GroEL.…”