Crystal structure of a chaperone-bound assembly intermediate of form I Rubisco

Abstract

The form I Rubisco of autotrophic bacteria, algae and plants is a complex of eight large (RbcL) and eight small (RbcS) subunits. It fixes atmospheric CO(2) in the dark reaction of photosynthesis. As shown for the cyanobacterial enzyme, folding of the RbcL subunits is mediated by the GroEL-GroES chaperonin system, and assembly requires the specialized chaperone RbcX, a homodimer of ~15-kDa subunits. Here we present the 3.2-Å crystal structure of a Rubisco assembly intermediate, consisting of the RbcL(8) core with eight RbcX(2) molecules bound. The structure reveals the molecular mechanism by which RbcX(2) mediates oligomeric assembly. Specifically, RbcX(2) provides positional information for proper formation of antiparallel RbcL dimers, thereby preventing RbcL-RbcL misalignment and off-pathway aggregation. The RbcL(8)(RbcX(2))(8) structure also suggests that RbcS functions by stabilizing the '60s loop' of RbcL in the catalytically active conformation.

Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in <i>Escherichia coli</i>

“…Therefore, residues in Sections 1, 2, 5 and 10 of RbcL have generally been ascribed other roles including catalysis Duff et al, 2000;Spreitzer, 2004, 2008;Spreitzer et al, 2005), RNA-binding translational arrest (Cohen et al, 2006) and Rubisco activase interaction (Ott et al, 2000). Interestingly, the C-terminal tail, which encompasses Section 10, was recently shown to interact with the RbcX Rubisco chaperone (Saschenbrecker et al, 2007;Bracher et al, 2011).…”

“…2) that gain entry into the chaperone might still misfold, condemning the mutant protein to a perpetual cycle of misfolding and unfolding within the chaperone until its degradation or aggregation in vivo (reviewed in Lin and Rye, 2006). Moreover, RbcL chimeras that are successfully folded by GroEL-GroES might still fail to assemble as complete holoenzymes in the absence of interaction with other Rubisco assemblyassisting proteins (Cloney et al, 1993;reviewed in Gutteridge and Gatenby, 1995;Saschenbrecker et al, 2007;Bracher et al, 2011;Kolesinski et al, 2014).…”

Role of Small Subunit in Mediating Assembly of Red-type Form I Rubisco

“…Our reconstitution experiments did not provide evidence that red-type RbcS first forms a stable tetramer platform on to which RbcL dimers assemble. Neither do the red-type RbcL subunits assemble independent of RbcS to stable octameric core complexes, in contrast to green-type cyanobacterial RbcL (20,21). Our data indicate that upon GroEL/ ES-mediated folding, the RbcL subunits form a range of soluble oligomeric states which are in dynamic equilibrium (Fig.…”

“…RbcS binding then results in a conformational change in the RbcL subunits, thereby causing the displacement of RbcX (21). In the case of RsRubisco, the RsRbcS subunits might play a similar role in assembly by linking RsRbcL dimers.…”

Molecular Chaperone GroEL – toward a Nano Toolkit in Protein Engineering, Production and Pharmacy

“…Yet another interesting spin is studying structures of GroEL/GroELS by a variety of techniques [4][5][6]15] and applying these approaches to studying protein and peptide structures in a bound state with GroEL by X-ray, NMR and cryo-electron microscopy [16][17][18][19][20].…”