Structural insights into the shape and assembly of photosynthetic GAPDH/CP12/PRK complex by small angle X-ray scattering

Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form together with the scaffold protein CP12, a supramolecular ternary complex of 498 kDa with stoichiometry [(GAPDH)-(CP12)-(PRK)]2. CP12 is an ubiquitous regulatory protein of oxygenic phototrophs that contains, with few exceptions, four conserved cysteines able to form two consecutive disulfide bridges. In higher plants as Arabidopsis thaliana, CP12 is predicted to be an intrinsically disordered protein (IDPs). 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. It is proposed that GAPDH/CP12/PRK supramolecular complex occurs in chloroplasts in the dark to ensure strong down-regulation of the Calvin cycle. Thus, the determination of the ternary complex structure is crucial for the understanding of the photosynthetic metabolism in light/dark regime. Crystallization trials to produce single crystals of the complex for X-ray diffraction experiments, failed. A structural study in solution by small angle scattering was then approached. The scattering profiles of the complex as well as of the PRK dimer were measured on the BM29, the dedicated bioSAXS beamline at the European Synchrotron Radiation Facility (Grenoble) and the ATSAS package was used for data analysis and modeling. First, the ab-initio shape of the PRK dimer was recovered using the program GASBOR. This bent-prolate structural model was then used together with the GAPDH-(CP12)2 complex crystallographic coordinates in the rigid-body modeling of the ternary complex against the experimental scattering curve performed with the program SASREFmx. The known stoichiometry of the complex was confirmed by the optimal data fitting. From the sorting of a big number of models obtained after multiple runs of the minimization procedure, an overall highly reproducible assembly emerged. The two GAPDH tetramers were in close contact and the two PRK dimers, both oriented with the concavity facing the centre of the complex, bridged them by interacting with the GAPDH-bound CP12s through the end regions. This rigid-body model of the complex was also consistent with previously reported hydrodynamic data. The SAXS-recovered structure is compatible with the present knowledge about this protein complex and highlights the propensity of GAPDH tetramers to interact reciprocally and associate in higher molecular weight forms as already reported from in vitro and in vivo observations.