The most prevalent form of the Rubisco enzyme is a complex of eight catalytic large subunits (RbcL) and eight regulatory small subunits (RbcS). Rubisco biogenesis depends on the assistance by specific molecular chaperones. The assembly chaperone RbcX stabilizes the RbcL subunits after folding by chaperonin and mediates their assembly to the RbcL8 core complex, from which RbcX is displaced by RbcS to form active holoenzyme. Two isoforms of RbcX are found in eukaryotes, RbcX-I, which is more closely related to cyanobacterial RbcX, and the more distant RbcX-II. The green algae Chlamydomonas reinhardtii contains only RbcX-II isoforms, CrRbcX-IIa and CrRbcX-IIb. Here we solved the crystal structure of CrRbcX-IIa and show that it forms an arc-shaped dimer with a central hydrophobic cleft for binding the C-terminal sequence of RbcL. Like other RbcX proteins, CrRbcX-IIa supports the assembly of cyanobacterial Rubisco in vitro, albeit with reduced activity relative to cyanobacterial RbcX-I. Structural analysis of a fusion protein of CrRbcX-IIa and the C-terminal peptide of RbcL suggests that the peptide binding mode of RbcX-II may differ from that of cyanobacterial RbcX. RbcX homologs appear to have adapted to their cognate Rubisco clients as a result of co-evolution.

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 (ɸEL) from the phage EL of Pseudomonas aeruginosa. ɸEL, 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, ɸEL failed to refold the encapsulation-dependent model substrate rhodanese and did not interact with E. coli GroES, the lid-shaped co-chaperone of GroEL. ɸEL forms tetradecameric double-ring complexes, which dissociate into single rings in the presence of ATP. Crystal structures of ɸEL (at 3.54 and 4.03 Å) 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-ɸEL revealed a double-ring structure composed of rings in the asymmetric state (3.45 Å 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, ɸEL may represent an evolutionarily more ancient chaperonin prior to acquisition of the encapsulation mechanism.