Molecular basis for the assembly of RuBisCO assisted by the chaperone Raf1

The folding and assembly of RuBisCO, the most abundant enzyme in nature, needs a series of chaperones, including the RuBisCO accumulation factor Raf1, which is highly conserved in cyanobacteria and plants. Here, we report the crystal structures of Raf1 from cyanobacteria Anabaena sp. PCC 7120 and its complex with RuBisCO large subunit RbcL. Structural analyses and biochemical assays reveal that each Raf1 dimer captures an RbcL dimer, with the C-terminal tail inserting into the catalytic pocket, and further mediates the assembly of RbcL dimers to form the octameric core of RuBisCO. Furthermore, the cryo-electron microscopy structures of the RbcL–Raf1–RbcS assembly intermediates enable us to see a dynamic assembly process from RbcL 8 Raf1 8 to the holoenzyme RbcL 8 RbcS 8 . In vitro assays also indicate that Raf1 can attenuate and reverse CcmM-mediated cyanobacterial RuBisCO condensation. Combined with previous findings, we propose a putative model for the assembly of cyanobacterial RuBisCO coordinated by the chaperone Raf1. RuBisCO is believed to be the most abundant enzyme on earth, and catalyses a critical step in carbon fixation. Assisted by different assembly factors, including Raf1, RuBisCO assembles into the holoenzyme comprising eight large subunits (RbcL) and eight small subunits (RbcS). Here, the researchers resolve high-resolution protein structures of the cyanobacterial RuBisCO octameric complex that reflect different steps of its assembly.