Coupled chaperone action in folding and assembly of hexadecameric Rubisco

Abstract

Form I Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), a complex of eight large (RbcL) and eight small (RbcS) subunits, catalyses the fixation of atmospheric CO(2) in photosynthesis. The limited catalytic efficiency of Rubisco has sparked extensive efforts to re-engineer the enzyme with the goal of enhancing agricultural productivity. To facilitate such efforts we analysed the formation of cyanobacterial form I Rubisco by in vitro reconstitution and cryo-electron microscopy. We show that RbcL subunit folding by the GroEL/GroES chaperonin is tightly coupled with assembly mediated by the chaperone RbcX(2). RbcL monomers remain partially unstable and retain high affinity for GroEL until captured by RbcX(2). As revealed by the structure of a RbcL(8)-(RbcX(2))(8) assembly intermediate, RbcX(2) acts as a molecular staple in stabilizing the RbcL subunits as dimers and facilitates RbcL(8) core assembly. Finally, addition of RbcS results in RbcX(2) release and holoenzyme formation. Specific assembly chaperones may be required more generally in the formation of complex oligomeric structures when folding is closely coupled to assembly.

Firefly luciferase mutants as sensors of proteome stress

“…Chaperones, transiently and non-covalently, shield the exposed hydrophobic surfaces on their substrate clients and prevent their non-specific interactions with other molecules. These proteins, in some cases, can also facilitate the post-translational assembly of polypeptides into oligomeric structures as in the assembly of the functional enzyme Ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) (Hemmingsen et al, 1988;Liu et al, 2010;Saschenbrecker et al, 2007). Many of the chaperones promote de novo folding by iterative cycles of substrate binding and release driven by ATP hydrolysis and may require co-factors or co-chaperones.…”

Assembly chaperones: a perspective

“…The simplest assumption is that additional chaperones may be required for the assembly of form I Rubisco in some species, including species of green plants, and recently, support has been gained for this idea from studies on the in vitro assembly of a cyanobacterial Rubisco after release of large subunits from GroEL. These studies suggest that an additional chaperone called RbcX is required for the assembly of cyanobacterial Rubisco in some species [35].…”

“…illustrates the complementary roles of the chaperonins and RbcX in the folding and assembly of the form I Rubisco from cyanobacteria, as deduced from in vitro renaturation studies with purified components [35]. Homologues of RbcX are found in plants [43], so an obvious strategy for future research on the chloroplast Rubiscos is to see whether they can also be renatured in vitro by supplying cognate chaperonins and RbcX.…”

Enhancing C3 Photosynthesis

“…The inability to assemble Rubisco from any photosynthetic eukaryote within Escherichia coli has hampered structure-function studies of higher plant Rubisco. Progress is being made in understanding chaperoning action in the folding and assembly of hexadecameric Rubisco (Liu et al, 2010). Although crystal structures of Rubisco are available, the possibility of improving the kinetic properties of Rubisco by rational design remains a goal for the future.…”