Insights into eukaryotic Rubisco assembly — Crystal structures of RbcX chaperones from Arabidopsis thaliana Piotr Kolesinski a , Przemyslaw Golik b , Przemyslaw Grudnik b , Janusz Piechota a , Michal Markiewicz c , Miroslaw Tarnawski a , Grzegorz Dubin b, d , Andrzej Szczepaniak a, ⁎ a Laboratory of Biophysics, Faculty of Biotechnology, University of Wroclaw, Przybyszewskiego 63/77, 51-148 Wroclaw, Poland b Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland c Department of Computational Biophysics and Bioinformatics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland d Malopolska Centre of Biotechnology, Gronostajowa 7a, 30-387, Krakow, Poland abstract article info Article history: Received 7 September 2012 Received in revised form 21 December 2012 Accepted 26 December 2012 Available online 4 January 2013 Keywords: RbcX Rubisco assembly Arabidopsis thaliana Background: Chloroplasts were formed by uptake of cyanobacteria into eukaryotic cells ca. 1.6 billion 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) dem- onstrate different affinities for the C-terminus of Rubisco large subunit and determine their crystal structures. Methods: 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 simu- lation. Results: 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. General significance: The presented results are the approach to elucidate the role of RbcX proteins in Rubisco assembly in higher plants. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Expansion of photosynthetic microorganisms led to the formation of an oxygenic atmosphere ca. 2.45 billion years ago [1,2]. As a result, form II of Rubisco was no longer capable of carbon dioxide fixation and gradually vanished (although it is still present in some lineages: the purple non-sulfur bacteria and eukaryotic dinoflagellates) [3]. In response to these changing environmental conditions, form I of Rubisco has emerged. In contrast to its evolutionarily older counterpart, form I conducts carboxylation in the oxygen rich environment [4]. The clear adaptive advantage is counterbalanced by a complicated assembly pathway. Form II of Rubisco folds into the active state with contribution of bacterial chaperonin GroEL/GroES only, whereas synthesis and as- sembly of form I require additional factors. Form I is a hexadecameric complex composed of eight large subunits (RbcL) forming the core of the protein which is capped by four small subunits (RbcS) from the top and another four small subunits form the bottom [3]. Adjustments at the active site of RbcL associated with evolution of form II into form I include significant structural rearrangement in the C-terminal domain. This particular region remains partially disordered after processing in- side the GroEL/GroES cage. As such, the C-terminal part exhibits high affinity for GroEL/GroES until it is captured by a second chaperone, the RbcX dimer. RbcX stabilizes the C-terminal domain of RbcL and facilitates RbcL dimer formation by maintaining appropriate orientation of both monomers [5]. It is believed that further stages of holoenzyme as- sembly occur spontaneously. Four dimers form an octameric core of the large Rubisco subunit. Subsequently, eight small subunits attach con- comitantly with RbcX chaperone dissociation completing the assembly process. The crystal structures of several prokaryotic RbcX proteins in a free (not RbcL associated) form have been determined previously [6–8]. Moreover, structural details of RbcL–RbcX interaction in prokaryotes have also been characterized [5]. The RbcX assembly chaperone is a Biochimica et Biophysica Acta 1830 (2013) 2899–2906 ⁎ Corresponding author. Tel.: +48 713756236; fax: +48 713756234. E-mail address: andrzej.szczepaniak@ibmb.uni.wroc.pl (A. Szczepaniak). 0304-4165/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagen.2012.12.025 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen