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 activit y of an isolated single seven-membered ring of the 14-mer. A known single-r ing mutant, GroEL(SR1), contains four point mutations that prevent the form ation of double-rings. That heptameric complex is functionally inactive bec ause 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 api cal domains of GroEL, appears to function by weakening the binding of GroES , without destabilizing the over all structure of GroEL44 mutant. We introd uced, 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 activ e. The new single-ring mutant, GroEL(SR44), was indeed effective in refoldi ng both heat and dithiothreitol-denatured mitochondrial malate dehydrogenas e with great efficiency. Further, unlike all smaller constructs of GroEL, t he expression of GroEL(SR44) in E. coli that contained no endogenous GroEL restored biological viability, but not as efficiently as does wild-type Gro EL. We envisage the notional evolution of the structure and properties of G roEL. The minichaperone core acts as a primitive chaperone by providing a b inding surface for denatured states that prevents their self-aggregation. T he assembly of seven minichaperones into a ring then enhances substrate bin ding by introducing avidity. The acquisition of binding sites for Am then a llows the modulation of substrate binding by introducing the allosteric mec hanism that causes cycling between strong and weak binding sites. This is a ccompanied by the acquisition by the heptamer of the binding of GroES, whic h functions as a lid to the central cavity and competes for peptide binding sites. Finally, dimerization of the heptamer enhances its biological activ ity. (C) 2000 Academic Press.