Creating the Functional Single-Ring GroEL-GroES Chaperonin Systems via Modulating GroEL-GroES Interaction

Chaperonin and cochaperonin, represented by E. coli GroEL and GroES, are essential molecular chaperones for protein folding. The double-ring assembly of GroEL is required to function with GroES, and a single-ring GroEL variant GroEL^SR forms a stable complex with GroES, arresting the chaperoning reaction cycle. GroES I25 interacts with GroEL; however, mutations of I25 abolish GroES-GroEL interaction due to the seven-fold mutational amplification in heptameric GroES. To weaken GroEL^SR-GroES interaction in a controlled manner, we used groES ⁷, a gene linking seven copies of groES, to incorporate I25 mutations in selected GroES modules in GroES⁷. We generated GroES⁷ variants with different numbers of GroESI25A or GroESI25D modules and different arrangements of the mutated modules, and biochemically characterized their interactions with GroEL^SR. GroES⁷ variants with two mutated modules participated in GroEL^SR-mediated protein folding in vitro. GroES⁷ variants with two or three mutated modules collaborated with GroEL^SR to perform chaperone function in vivo: three GroES⁷ variants functioned with GroEL^SR under both normal and heat-shock conditions. Our studies on functional single-ring bacterial chaperonin systems are informative to the single-ring human mitochondrial chaperonin mtHsp60-mtHsp10, and will provide insights into how the double-ring bacterial system has evolved to the single-ring mtHsp60-mtHsp10.