Mechanism of Enzyme Repair by the AAA+ Chaperone Rubisco Activase

How AAA+ chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA+ protein Rubisco activase (Rca) in metabolic repair of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits containing eight catalytic sites. Rubisco is prone to inhibition by tight-binding sugar phosphates, whose removal is catalyzed by Rca. We engineered a stable Rca hexamer ring and analyzed its functional interaction with Rubisco. Hydrogen/deuterium exchange and chemical crosslinking showed that Rca structurally destabilizes elements of the Rubisco active site with remarkable selectivity. Cryo-electron microscopy revealed that Rca docks onto Rubisco over one active site at a time, positioning the C-terminal strand of RbcL, which stabilizes the catalytic center, for access to the Rca hexamer pore. The pulling force of Rca is fine-tuned to avoid global destabilization and allow for precise enzyme repair. [Display omitted] •Rca contacts both large (RbcL) and small (RbcS) subunits of inactive Rubisco•Rca docks onto Rubisco side-on, engaging the flexible C-terminal strand of RbcL•Rca conformationally repairs one Rubisco catalytic site at a time•Rca performs “molecular surgery” with minimal structural perturbation The key photosynthetic enzyme Rubisco undergoes inhibition by substrate and non-substrate sugar phosphates. Inhibited Rubisco requires metabolic repair by the AAA+ chaperone Rubisco activase. Using an integrated approach of biochemical and structural techniques, Bhat et al. show that activase repairs the defective enzyme with remarkable precision, avoiding global structural perturbation.