From minichaperone to GroEL 2: importance of avidity of the multisite ring structure

Structural studies on minichaperones and GroEL imply a continuous ring of binding sites around the neck of GroEL. To investigate the importance of this ring, we constructed an artificial heptameric assembly of minichaperones to mimic their arrangement in GroEL. The heptameric Gp31 co-chaperonin from bacteriophage T4, an analogue of GroES, was used as a scaffold to display the GroEL minichaperones. A fusion protein, MC(7), was generated by replacing a part of the highly mobile loop of Gp31 (residues 23-44) with the sequence of the minichaperone (residues 191-376 of GroEL). The purified recombinant protein assembled into a heptameric ring composed of seven 30.6 kDa subunits. Although single minichaperones (residues 193-335 to 191-376 of GroEL) have certain chaperone activities in vitro and in vivo, they cannot refold heat and dithiothreitol-denatured mitochondrial malate dehydrogenase (mtMDH), a reaction that normally requires GroEL, its co-chaperonin GroES and ATP. But, MC(7) refolded MDH in vitro. The expression of MC(7) complements in vivo two temperature-sensitive Escherichia coli alleles, groEL44 and groEL673, at 43 degrees C. Although MC(7) could not compensate for the complete absence of GroEL in vivo, it enhanced the colony-forming ability of cells containing limiting amounts of wild-type GroEL at 37 degrees C. MC(7 )also reduces aggregate formation and cell death in mammalian cell models of Huntington's disease. The assembly of seven minichaperone subunits on a heptameric ring significantly improves their activity, demonstrating the importance of avidity in GroEL function.