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Mechanism of Rubisco repair by the AAA+ chaperone Rubisco activase All plants and some cyanobacteria depend on the hexameric AAA+ chaperone Rubisco activase to repair Rubisco s CO2 fixation function. Structural analysis by crystallography and cryo-EM indicates that cyanobacterial Rubisco activase accomplishes this task by destabilising the layered lid above the active site pocket, leading to the release of inhibitory sugar. Ribulose-1,5-bisphosphate carboxylase/oxygenase, Rubisco, is a hexadecameric complex of eight large (RbcL) and eight small (RbcS) subunits. Photosynthetic organisms use Rubisco to catalyse the carboxylation of the 5-carbon sugar substrate ribulose-1,5-bisphosphate in the Calvin- Benson-Bassham cycle. Rubisco is prone to inactivation by tight-binding inhibitory sugar phosphates. To repair

the inhibited Rubisco, plants express the hexameric AAA+ (ATPases associated with diverse cellular activities) chaperone Rubisco activase (Rca). The subunits of AAA+ enzymes undergo ATP-regulated conformational cycling, resulting in the insertion of substrate polypeptide into the central pore. How plant Rca repairs the inhibited Rubisco had been a puzzling problem. A distantly related Rca of the proteobacterium Rhodobacter sphaeroides was recently shown to pull at the extended C-terminal tail residues of the RbcL subunit of its cognate Rubisco [1,2]. However, these additional residues are absent in the RbcL subunits of plants and cyanobacteria.

This work studied the Rca protein from the b-cyanobacterium Nostoc PCC7120 (Nos) because its AAA+ core has high sequence similarity to that of plant Rca (Figure 35a). The crystal structure of a C-terminally truncated hexameric NosRca (NosRcaΔC) with ADP bound to alternating subunits (Figure 35b) was solved. NosRca lacks the N-domain of plant Rca but instead contains a domain at the C-terminus that is similar to the small subunit of Rubisco called SSUL (small subunit-like) (Figure 35a). Such SSUL domains are also found in the protein CcmM of b-cyanobacteria. A crystal structure of the SSUL domain of NosRca solved by multi-wavelength anomalous diffraction (MAD) at beamline BM30A and determined at atomic resolution at beamline ID30A-1 showed similarity with the SSUL domain of CcmM from Synechococcus elongatus PCC7942 (Se) (Figure 35c) [3]. In b-cyanobacteria, both Rubisco and Rca are encapsulated into carboxysomes, microcompartments with a proteinaceous shell. Since it was recently shown that SSUL domains of CcmM bind to Rubisco, resulting in the formation of a liquid-liquid phase- separated (LLPS) condensate [3], this finding suggested that the SSUL domains of hexameric NosRca also interact with Rubisco, and thereby would serve to recruit Rca into the pre-carboxysome together with Rubisco and CcmM. Indeed, it was found that the recruitment of NosRca into a LLPS condensate with Rubisco is dependent on the presence of its SSUL domains.

Cryo-EM was used to elucidate the structural basis for Rubisco reactivation. A stable complex of NosRcaΔC-Rubisco was generated by mixing inhibitor-bound Rubisco with NosRcaΔC in the presence of ATP to initiate the interaction. After 10s, the repair function of NosRcaΔC was halted by the addition of the slowly hydrolysing ATP analogue ATPγS. Cryo-EM analysis and single-particle reconstruction

Fig. 35: a) Comparison of the domain structures of the eukaryotic Rca from N. tabacum (NtRca) and the prokaryotic Rca from Nostoc PCC7120 (NosRca). b) NosRcaΔC hexamer (PDB: 6Z1E). cis, surface from which the linkers to the SSUL domains emanate; trans, the opposite surface to cis. c) Comparison of the SSUL domain of NosRca (left; PDB: 6HAS) with SSUL1 domain of the CcmM protein from S. elongatus PCC 7942 (Se) (right; PDB: 6HBA).