Molecular Properties of Chloroplastic CP12 and its role in the Assembling of a Supramolecular Complex of Calvin Cycle Enzymes

CP12 is a small regulatory peptide localized in chloroplast stroma, where it leads to the assembly of a supramolecular complex with glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK). Once embodied in the complex, both enzymes alter their kinetic properties according to the general regulation of photosynthetic carbon assimilation in land plants (dark-inactivation). CP12–2 (isoform 2 of Arabidopsis thaliana) lacks a defined three-dimensional structure and can thus be regarded as an intrinsically unstructured proteins (IUP). However, CP12–2 contains four cysteines which can form two intramolecular disulfide bridges with different midpoint redox potentials (Em,7.9 = −326 ± 2 and −352 ± 6 mV). Dynamic light scattering analysis demonstrated that CP12-2 is a monomer of 9 kDa that under oxidizing condition interacts with A4-GAPDH complexed with NAD, leading to the formation of a 170 kDa binary complex (A4-GAPDH)-(CP12- 2)2. Oxidized PRK (85 kDa) binds to this binary complex giving rise to a ternary complex of 498 kDa, consistent with a [(A4-GAPDH)-(CP12- 2)2–(PRK)]2 stoichiometry. Site specific mutants suggest that the C-terminal disulfide of CP12 is responsible for the interaction with A4-GAPDH, while the N-terminal disulfide is involved in the interaction with PRK.