From minichaperone to GroEL 3: properties of an active single-ring mutant of GroEL.

The next step in our reductional analysis of GroEL was to study the activity of an isolated single seven-membered ring of the 14-mer. A known single-ring mutant, GroEL(SR1), contains four point mutations that prevent the formation of double-rings. That heptameric complex is functionally inactive because it is unable to release GroES. We found that the mutation E191G, which is responsible for the temperature sensitive (ts) Escherichia coli allele groEL44 and is located in the hinge region between the intermediate and apical domains of GroEL, appears to function by weakening the binding of GroES, without destabilizing the overall structure of GroEL44 mutant. We introduced, therefore, the mutation E191G into GroEL(SR1) in order to generate a single-ring mutant that may have weaker binding of GroES and hence be active. The new single-ring mutant, GroEL(SR44), was indeed effective in refolding both heat and dithiothreitol-denatured mitochondrial malate dehydrogenase with great efficiency. Further, unlike all smaller constructs of GroEL, the expression of GroEL(SR44) in E. coli that contained no endogenous GroEL restored biological viability, but not as efficiently as does wild-type GroEL. We envisage the notional evolution of the structure and properties of GroEL. The minichaperone core acts as a primitive chaperone by providing a binding surface for denatured states that prevents their self-aggregation. The assembly of seven minichaperones into a ring then enhances substrate binding by introducing avidity. The acquisition of binding sites for ATP then allows the modulation of substrate binding by introducing the allosteric mechanism that causes cycling between strong and weak binding sites. This is accompanied by the acquisition by the heptamer of the binding of GroES, which functions as a lid to the central cavity and competes for peptide binding sites. Finally, dimerization of the heptamer enhances its biological activity.