Bracher, A. | bioRxiv Science

bioRxiv2020

Dual Role of a Rubisco Activase in Metabolic Repair and Carboxysome Organization

Rubisco, the key enzyme of CO2 fixation in photosynthesis, is prone to inactivation by inhibitory sugar phosphates. Inhibited Rubisco undergoes conformational repair by the hexameric AAA+ chaperone Rubisco activase (Rca) in a process that is not well understood. Here we performed a structural and mechanistic analysis of cyanobacterial Rca, a close homolog of plant Rca. In the Rca:Rubisco complex, Rca is positioned over the Rubisco catalytic site under repair and pulls the N-terminal tail of the large Rubisco subunit (RbcL) into the hexamer pore. Simultaneous displacement of the C-terminus of the adjacent RbcL opens the catalytic site for inhibitor release. An alternative interaction of Rca with Rubisco is mediated by C-terminal domains that resemble the small Rubisco subunit. These domains, together with the N-terminal AAA+ hexamer, ensure that Rca is packaged with Rubisco into carboxysomes. Cyanobacterial Rca is a dual-purpose protein with functions in Rubisco repair and carboxysome organization.

biochemistry↗

bioRxiv2020

Structure and conformational cycle of a bacteriophage-encoded chaperonin

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 evolutionary 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 [A]) 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 [A] 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 evolutionary more ancient chaperonin prior to acquisition of the encapsulation mechanism.

biochemistry↗