Oligomerization of an archaeal group II chaperonin is mediated by N-terminal salt bridges.

Group II chaperonins (Cpns) are essential mediators of cellular protein folding in eukaryotes and archaea. They consist of two back-to-back rings forming symmetrical cavities in which non-native substrates undergo appropriate folding, but the primary structural basis for the double ring formation remains unclear. To address this, we carried out systematic mutagenesis on the Cpn from the hyperthermophilic archaeon Pyrococcus furiosus, which is assembled from identical subunits. In our study, (21)GRDAQRMNIL(30) was found to be a critical domain for double ring formation. Deletion of this section stepwise beyond residue 20 resulted in failure to assemble double-ring oligomers and the progressive loss of chaperone function. A key domain spanning the residues 21-50 that is essential for the formation of tetramers that appear to be the intermediates for double ring assembly. Mutation of either Arg22 to Ala22 or Glu37 to Ala37 resulted in similar defects in double-ring assembly and functional deficits. A mutant with Arg22 and Glu37 switched assembled double rings efficiently and exhibited chaperone functions similar to the wild-type. Therefore, Arg22 and Glu37 could form inter-ring salt bridges critical for double ring formation. In addition, Asn28 and Ile29 were found to contribute significantly to ring formation. Sequence alignment revealed that these four residues are highly conserved among group II Cpns. This is the first report of a comprehensive N-terminal mutational analysis for elucidating the oligomerization of group II Cpns.