Significance of the N-terminal domain for the function of chloroplast cpn20 chaperonin

Chaperonins cpn60 and cpn10 are essential proteins involved in cellular protein folding. Plant chloroplasts contain a unique version of the cpn10 co-chaperonin, cpn20, which consists of two homologous cpn10-like domains (N-cpn20 and C-cpn20) that are connected by a short linker region. Although cpn20 seems to function like other single domain cpn10 oligomers, the structure and specific functions of the domains are not understood. We mutated amino acids in the "mobile loop" regions of N-cpn20, C-cpn20 or both: a highly conserved glycine, which was shown to be important for flexibility of the mobile loop, and a leucine residue shown to be involved in binding of co-chaperonin to chaperonin. The mutant proteins were purified and their oligomeric structure validated by gel filtration, native gel electrophoresis, and circular dichroism. Functional assays of protein refolding and inhibition of GroEL ATPase both showed (i) mutation of the conserved glycine reduced the activity of cpn20, whether in N-cpn20 (G32A) or C-cpn20 (G130A). The same mutation in the bacterial cpn10 (GroES G24A) had no effect on activity. (ii) Mutations in the highly conserved leucine of N-cpn20 (L35A) and in the corresponding L27A of GroES resulted in inactive protein. (iii) In contrast, mutant L133A, in which the conserved leucine of C-cpn20 was altered, retained 55% activity. We conclude that the structure of cpn20 is much more sensitive to alterations in the mobile loop than is the structure of GroES. Moreover, only N-cpn20 is necessary for activity of cpn20. However, full and efficient functioning requires both domains.