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Windhof, Amanda (2011): Rubisco folding and oligomeric assembly: Detailed analysis of an assembly intermediate. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

To become biologically active, a protein must fold into a distinct three-dimensional structure. Many non-native proteins require molecular chaperones to support folding and assembly. These molecular chaperones are important for de novo protein folding as well as refolding of denatured proteins under stress conditions. A certain subset of chaperones, the chaperonins, are required for the folding of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco); furthermore, correct folding of Rubisco is also aided by the Hsp70 chaperone system. Rubisco catalyzes the initial step of CO2 assimilation in the Calvin-Benson-Bassham (CBB) cycle. Unfortunately, this enzyme is extremely inefficient, not only does it exhibit a slow catalytic rate (three CO2 molecules fixed per second per Rubisco) but it also discriminates poorly between the assimilation of CO2 and O2 to its sugar-phosphate substrate ribulose-1,5-bisphosphate (RuBP), the latter resulting in loss of photosynthetic efficiency. Due to these inefficiencies, carbon fixation by Rubisco is the rate limiting step of the CBB cycle. Photosynthetic organisms must produce tremendous amounts of Rubisco to alleviate these shortcomings; therefore significant quantities of nitrogen stores are invested in the production of Rubisco making Rubisco the most abundant protein on earth. These drawbacks of Rubisco have important implications in increasing CO2 concentrations and temperatures in the context of global warming. The ability to engineer a more efficient Rubisco could potentially reduce photosynthetic water usage, increase plant growth yield, and reduce nitrogen usage is plants. However, eukaryotic Rubisco cannot fold and assemble outside of the chloroplast, hindering advancements in creating a more efficient Rubisco. Form I Rubisco, found in higher plants, algae, and cyanobacteria, is a hexadecameric complex consisting of a core of eight ~50 kDa large subunits (RbcL), which is capped by four ~15 kDa small subunits (RbcS) on each end. The discovery of a Rubisco-specific assembly chaperone, RbcX, has lead to a better understanding of the components necessary for the form I Rubisco assembly process. RbcX is a homodimer of ~15 kDa subunits consisting of four α- helices aligned in an anti-parallel fashion along the α4 helix. RbcX2 functions as a stabilizer of folded RbcL by recognizing a highly conserved C-terminal sequence of RbcL: EIKFEFD, termed the C-terminal recognition motif. As has been demonstrated by studies of cyanobacterial Rubisco, de novo synthesized RbcL is folded by the chaperonins, whereupon RbcX2 stabilizes the folded RbcL monomer upon release from the folding cavity and then assists in the formation of the RbcL8 core. RbcX2 forms a dynamic complex with RbcL8 and as a result, RbcX2 is readily displaced by RbcS docking in an ATP-independent manner, thereby creating the functional holoenzyme. However, the exact mechanism by which RbcS binding displaces RbcX2 from the RbcL8 core is still unknown. Furthermore, though much advancement has been made in the understanding of form I Rubisco folding and assembly, an exact and detailed mechanism of form I Rubisco assembly is still lacking. The highly dynamic complex of RbcL/RbcX is critical for the formation of the holoenzyme; however it has hindered attempts to characterize critical regions of RbcL that interact with the peripheral regions of RbcX2. An important observation arose when heterologous RbcL and RbcX2 components interacted; a stable complex could form enabling in depth characterization of the RbcL/RbcX2 interaction. In the present study, the detailed structural mechanism of RbcX2-mediated cyanobacterial form I Rubisco assembly is elucidated. To obtain molecular insight into the RbcX2-mediated assembly process of cyanobacterial form I Rubisco, cryo-EM and crystallographic studies in concert with mutational analysis were employed by taking advantage of the high affinity interaction between RbcL and RbcX2 in the heterologous system (Synechococcus sp. PCC6301 RbcL and Anabaena sp. CA RbcX2). Structure guided mutational analysis based on the 3.2 Å crystal structure of the RbcL8/(RbcX2)8 assembly intermediate were utilized to determine the precise interaction site between the body of RbcL and the peripheral region of RbcX2. From these studies a critical salt bridge could be identified that functions as a guidepoint for correct dimer formation, and it was observed that RbcX2 exclusively mediates Rubisco dimer assembly. Furthermore, the mechanism of RbcX2 displacement from the RbcL8 core by RbcS binding was elucidated as well as indications of how RbcS docking on the RbcL8 core is imperative for full form I Rubisco catalytic function by stabilizing the enzymatically competent conformation of an N-terminal loop of Rubisco termed the ‘60ies loop’. Finally, initial attempts in in vitro reconstitution of eukaryotic Rubisco are reported along with the characterization of Arabidopsis thaliana RbcX2 binding to the C-terminal recognition motif of the Rubisco large subunit from various species.