Structural insights into cyanobacterial RuBisCO assembly coordinated by two chaperones Raf1 and RbcX.

RuBisCO is the most abundant enzyme in nature, catalyzing the fixation of CO in photosynthesis. Its common form consists of eight RbcL and eight RbcS subunits, the assembly of which requires a series of chaperones that include RbcX and RuBisCO accumulation factor 1 (Raf1). To understand how these RuBisCO-specific chaperones function during cyanobacterial RbcLRbcS (LS) holoenzyme formation, we solved a 3.3-Å cryo-electron microscopy structure of a 32-subunit RbcLRaf1RbcX (LFX) assembly intermediate from Anabaena sp. PCC 7120. Comparison to the previously resolved LF and LX structures together with biochemical assays revealed that the LFX complex forms a rather dynamic structural intermediate, favoring RbcS displacement of Raf1 and RbcX. In vitro assays further demonstrated that both Raf1 and RbcX function to regulate RuBisCO condensate formation by restricting CcmM35 binding to the stably assembled LS holoenzymes. Combined with previous findings, we propose a model on how Raf1 and RbcX work in concert to facilitate, and regulate, cyanobacterial RuBisCO assembly as well as disassembly of RuBisCO condensates.