Archaeal group II chaperonin mediates protein folding in the cis-cavity without a detachable GroES-like co-chaperonin

Abstract

Group II chaperonins of archaea and eukaryotes are distinct from group I chaperonins of bacteria. Whereas group I chaperonins require the co-chaperonin Cpn-10 or GroES for protein folding, no co-chaperonin has been known for group II. The protein folding mechanism of group II chaperonins is not yet clear. To understand this mechanism, we examined protein refolding by the recombinant α or β-subunit chaperonin homo-oligomer (α16mer and β16mer) from a hyperthermoplilic archaeum, Thermococcus strain KS-1, using a model substrate, green fluorescent protein (GFP). The α16mer and β16mer captured the non-native GFP and promoted its refolding without any co-chaperonin in an ATP dependent manner. A non-hydrolyzable ATP analog, AMP-PNP, induced the GFP refolding mediated by β16mer but not by the α16mer. A mutant α-subunit chaperonin homo-oligomer (trap-α) could capture the non-native protein but lacked the ability to refold it. Although trap-α suppressed ATP-dependent refolding of GFP mediated by α16mer or β16mer, it did not affect the AMP-PNP-dependent refolding. This indicated that the GFP refolding mediated by β16mer with AMP-PNP was not accessible to the trap-α. Gel filtration chromatography and a protease protection experiment revealed that this refolded GFP, in the presence of AMP-PNP, was associated with β16mer. After the completion of GFP refolding mediated by β16mer with AMP-PNP, addition of ATP induced an additional refolding of GFP. Furthermore, the β16mer preincubated with AMP-PNP showed the ability to capture the non-native GFP. These suggest that AMP-PNP induced one of two chaperonin rings (cis-ring) to close and induced protein refolding in this ring, and that the other ring (trans-ring) could capture the unfolded GFP which was refolded by adding ATP. The present data indicate that, in the group II chaperonin of Thermococcus strain KS-1, the protein folding proceeds in its cis-ring in an ATP-dependent fashion without any co-chaperonin.