Insights into eukaryotic Rubisco assembly — Crystal structures of RbcX chaperones from Arabidopsis thaliana

• Published in: Biochimica et biophysica acta Vol. 1830; no. 4; pp. 2899 - 2906
• Main Authors: Kolesinski, Piotr, Golik, Przemyslaw, Grudnik, Przemyslaw, Piechota, Janusz, Markiewicz, Michał, Tarnawski, Miroslaw, Dubin, Grzegorz, Szczepaniak, Andrzej
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2013
• Subjects: Amino Acid Sequence; amino acid sequences; Arabidopsis Proteins - chemistry; Arabidopsis thaliana; biosynthesis; Chloroplast Proteins - chemistry; chloroplasts; crystal structure; Crystallization; genes; molecular chaperones; Molecular Chaperones - chemistry; Molecular Sequence Data; nuclear genome; Protein Conformation; Protein Multimerization; Protein Stability; RbcX; ribulose-bisphosphate carboxylase; Ribulose-Bisphosphate Carboxylase - chemistry; Rubisco assembly; sequence homology; titration; Arabidopsis thaliana; Rubisco assembly; RbcX
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2012.12.025

Chloroplasts were formed by uptake of cyanobacteria into eukaryotic cells ca. 1.6billion years ago. During evolution most of the cyanobacterial genes were transferred from the chloroplast to the nuclear genome. The rbcX gene, encoding an assembly chaperone required for Rubisco biosynthesis in cyanobacteria, was duplicated. Here we demonstrate that homologous eukaryotic chaperones (AtRbcX1 and AtRbcX2) demonstrate different affinities for the C-terminus of Rubisco large subunit and determine their crystal structures. Three-dimensional structures of AtRbcX1 and AtRbcX2 were resolved by the molecular replacement method. Equilibrium binding constants of the C-terminal RbcL peptide by AtRbcX proteins were determined by spectrofluorimetric titration. The binding mode of RbcX–RbcL was predicted using molecular dynamic simulation. We provide crystal structures of both chaperones from Arabidopsis thaliana providing the first structural insight into Rubisco assembly chaperones form higher plants. Despite the low sequence homology of eukaryotic and cyanobacterial Rubisco chaperones the eukaryotic counterparts exhibit surprisingly high similarity of the overall fold to previously determined prokaryotic structures. Modeling studies demonstrate that the overall mode of the binding of RbcL peptide is conserved among these proteins. As such, the evolution of RbcX chaperones is another example of maintaining conserved structural features despite significant drift in the primary amino acid sequence. The presented results are the approach to elucidate the role of RbcX proteins in Rubisco assembly in higher plants. ► Structural details of eucaryotic Rubisco chaperones are shown for the first time. ► Crystal structures of two RbcX homologs from Arabidopsis thaliana are provided. ► Both AtRbcX share the same overall topology with their cyanobacterial equivalents. ► Structural details differ significantly in the case of AtRbcX1. ► High conservation of the binding pockets of distant RbcX chaperones is revealed.