Spontaneous assembly of photosynthetic supramolecular complexes as mediated by the intrinsically unstructured protein CP12.
CP12 is a protein of 8.7 kDa that contributes to Calvin cycle regulation by acting as a scaffold element in the formation of a supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) in photosynthetic organisms. NMR studies of recombinant CP12 (isoform 2) of Arabidopsis thaliana show that CP12-2 is poorly structured. CP12-2 is monomeric in solution and contains four cysteines, which can form two intramolecular disulfides with midpoint redox potentials of -326 and -352 mV, respectively, at pH 7.9. Site-specific mutants indicate that the C-terminal disulfide is involved in the interaction between CP12-2 and GAPDH (isoform A(4)), whereas the N-terminal disulfide is involved in the interaction between this binary complex and PRK. In the presence of NAD, oxidized CP12-2 interacts with A(4)-GAPDH (K(D) = 0.18 microm) to form a binary complex of 170 kDa with (A(4)-GAPDH)-(CP12-2)(2) stoichiometry, as determined by isothermal titration calorimetry and multiangle light scattering analysis. PRK is a dimer and by interacting with this binary complex (K(D) = 0.17 microm) leads to a 498-kDa ternary complex constituted by two binary complexes and two PRK dimers, i.e. ((A(4)-GAPDH)-(CP12-2)(2)-(PRK))(2). Thermodynamic parameters indicate that assembly of both binary and ternary complexes is exoergonic although penalized by a decrease in entropy that suggests an induced folding of CP12-2 upon binding to partner proteins. The redox dependence of events leading to supramolecular complexes is consistent with a role of CP12 in coordinating the reversible inactivation of chloroplast enzymes A(4)-GAPDH and PRK during darkness in photosynthetic tissues.
Marri
Lucia
L
Laboratory of Molecular Plant Physiology, Department of Experimental Evolutionary Biology, University of Bologna, Via Irnerio 42, Bologna 40126, Italy.
Trost
Paolo
P
Trivelli
Xavier
X
Gonnelli
Leonardo
L
Pupillo
Paolo
P
Sparla
Francesca
F
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D010788:Photosynthesis / Q000502:physiology*; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics
D029681:Arabidopsis Proteins; D007527:Isoenzymes; D045322:Photosynthetic Reaction Center Complex Proteins; D010940:Plant Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase