Structural Analysis of the Rubisco-Assembly Chaperone RbcX-II from Chlamydomonas reinhardtii

Abstract

The most prevalent form of the Rubisco enzyme is a complex of eight catalytic large subunits (RbcL) and eight regulatory small subunits (RbcS). Rubisco biogenesis depends on the assistance by specific molecular chaperones. The assembly chaperone RbcX stabilizes the RbcL subunits after folding by chaperonin and mediates their assembly to the RbcL8 core complex, from which RbcX is displaced by RbcS to form active holoenzyme. Two isoforms of RbcX are found in eukaryotes, RbcX-I, which is more closely related to cyanobacterial RbcX, and the more distant RbcX-II. The green algae Chlamydomonas reinhardtii contains only RbcX-II isoforms, CrRbcX-IIa and CrRbcX-IIb. Here we solved the crystal structure of CrRbcX-IIa and show that it forms an arc-shaped dimer with a central hydrophobic cleft for binding the C-terminal sequence of RbcL. Like other RbcX proteins, CrRbcX-IIa supports the assembly of cyanobacterial Rubisco in vitro, albeit with reduced activity relative to cyanobacterial RbcX-I. Structural analysis of a fusion protein of CrRbcX-IIa and the C-terminal peptide of RbcL suggests that the peptide binding mode of RbcX-II may differ from that of cyanobacterial RbcX. RbcX homologs appear to have adapted to their cognate Rubisco clients as a result of co-evolution.

Rubisco Assembly in the Chloroplast

“…RbcX is conserved from the cyanobacteria to plants (Hauser et al, 2015b ). Co-expression of the RbcX genes from various cyanobacteria as well as from C. reinhartii or A. thaliana , was shown to enhance the assembly of cyanobacterial Rubisco in E. coli (Li and Tabita, 1997 ; Onizuka et al, 2004 ; Saschenbrecker et al, 2007 ; Kolesinski et al, 2011 ; Bracher et al, 2015 ), suggesting a conserved mode of function for all the homologs. Insertional inactivation of RbcX genes that were located in or outside of the Rubisco operons in two cyanobacteria strains, suggested that the RbcX protein may be essential for Rubisco biogenesis only when it is expressed from the Rubisco operon (Li and Tabita, 1997 ; Emlyn-Jones et al, 2006 ).…”

“…Interestingly, Chlamydomonas encodes only the AtRbcX1 homologs, CrRbcXA and CrRbcXB. CrRbcXA was structurally and functionally characterized and shown to support cyanobacterial Rubisco assembly (Bracher et al, 2015 ). In the future, characterization of RbcX mutants as well as additional biochemical studies could reveal their precise role in Rubisco assembly and the unique properties of each homolog.…”

RAF2 is a RuBisCO assembly factor in Arabidopsis thaliana

“…Multiple chaperones have evolved to stabilize RuBisCO LS dimers during the multi-step assembly process prior to SS incorporation. In b-cyanobacteria and land plants, RbcX prevents LS aggregation by stabilizing the unstructured large subunit before being displaced by SS, although RbcX is not required for RuBisCO function in all organisms that encode it (Bracher et al, 2015;Hauser et al, 2015b). RAF1 (RuBisCO Accumulation Factor 1), found in eukaryotes and certain cyanobacteria, operates similarly to RbcX in that it also stabilizes the LS dimer before SS incorporation (Feiz et al, 2012).…”

Evolutionary escape from the Rubisco activase requirement

“…Three assembly factors have been identified for green-type Rubisco; RbcX, Rubisco accumulation factor 1 (Raf1), and Raf2 (Bracher et al, 2015;Feiz et al, 2014;Feiz et al, 2012;Hauser et al, 2015a;Kolesinski et al, 2014;Li et al, 1997;Liu et al, 2010 interacts with RbcL downstream of chaperonin-associated RbcL folding. They function to stabilized RbcL2 but bind using different interaction sites (Bracher et al, 2017).…”