Insight into the assembly of the Calvin cycle regulatory GAPDH/CP12/PRK complex by SAXS

Photosynthetic organisms produce sugars through the Calvin-Benson cycle, consuming carbon dioxide and energy provided by the conversion of light to chemical energy. The smooth proceeding of photosynthesis is controlled by different regulatory systems including the transient formation of protein complexes. Through the scaffold protein CP12, which is predicted to be intrinsically disordered, two enzymes of the cycle, glyceraldehyde-3-phosphate dehydrogenase (tetrameric GAPDH) and phosphoribulokinase (dimeric PRK), are regulated by formation of a supramolecular ternary complex of 498 kDa with stoichiometry [GAPDH-(CP12)2-(PRK)]2. The activities of GAPDH and PRK enzymes are inhibited by complex formation and fully recovered upon dissociation of the complex at the onset of light, providing an effective means for regulation of the Calvin cycle in vivo.Small angle X-ray scattering analysis was performed on the pre-formed complex and its free components all from Arabidopsis thaliana, and the ATSAS package was used for data analysis and modelling. A concave bent and screwed ab-initio shape of the PRK dimer was recovered, while a combined rigid-body/dummy-residue model was obtained for the GAPDH-(CP12)2 binary complex in order to take into account a small rearrangement of the known crystallographic subunits positions and the missing CP12 amino acids. These models were then used in the rigid-body modelling of the ternary complex against the experimental scattering curve, allowing for partial dissociation. The known stoichiometry of the complex was confirmed and from the sorting of a big number of models obtained with multiple runs of the minimization procedure, an overall reproducible assembly emerged. The structure of the ternary complex appears more compact with respect to the previous pictorial models and the two GAPDHs proximity suggests an unsuspected involvement of an interaction between them in the overall complex stabilization.