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Abstract

Chaperonins are ubiquitous molecular chaperones found in all domains of life. They form ring-shaped complexes that assist in the folding of substrate proteins in an ATP-dependent reaction cycle. Key to the folding cycle is the transient encapsulation of substrate proteins by the chaperonin. Here we present a structural and functional characterization of the chaperonin gp146 (phi EL) from the phage EL of Pseudomonas aeruginosa. phi EL, an evolutionarily distant homolog of bacterial GroEL, is active in ATP hydrolysis and prevents the aggregation of denatured protein in a nucleotide-dependent manner. However, phi EL failed to refold the encapsulation-dependent model substrate rhodanese and did not interact with E. coli GroES, the lid-shaped co-chaperone of GroEL. phi EL forms tetradecameric double-ring complexes, which dissociate into single rings in the presence of ATP. Crystal structures of phi EL (at 3.54 and 4.03 angstrom) in presence of ATP.BeFx revealed two distinct single-ring conformational states, both with open access to the ring cavity. One state showed uniform ATP-bound subunit conformations (symmetric state), whereas the second combined distinct ATP- and ADP-bound subunit conformations (asymmetric state). Cryo-electron microscopy of apo-phi EL revealed a double-ring structure composed of rings in the asymmetric state (3.45 angstrom resolution). We propose that the phage chaperonin undergoes nucleotide-dependent conformational switching between double- and single rings and functions in aggregation prevention without substrate protein encapsulation. Thus, phi EL may represent an evolutionarily more ancient chaperonin prior to acquisition of the encapsulation mechanism.