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J. Biol. Chem., Vol. 283, Issue 4, 1831-1838, January 25, 2008

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Spontaneous Assembly of Photosynthetic Supramolecular Complexes as Mediated by the Intrinsically Unstructured Protein CP12^*

Lucia Marri, Paolo Trost¹, Xavier Trivelli, Leonardo Gonnelli^¶, Paolo Pupillo, and Francesca Sparla

From the Laboratory of Molecular Plant Physiology, Department of Experimental Evolutionary Biology, University of Bologna, Via Irnerio 42, Bologna 40126, Italy, the Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576 CNRS, IFR147, Science and Technology University of Lille, Villeneuve d'Ascq 59655, France, and the ^¶Magnetic Resonance Center, University of Florence, Via Luigi Sacconi 6, Sesto Fiorentino (Firenze) 50019, Italy

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 A₄), 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 A₄-GAPDH (K_D = 0.18 µM) to form a binary complex of 170 kDa with (A₄-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 µM) leads to a 498-kDa ternary complex constituted by two binary complexes and two PRK dimers, i.e. ((A₄-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 A₄-GAPDH and PRK during darkness in photosynthetic tissues.

Received for publication, July 10, 2007 , and in revised form, September 27, 2007.

^* This work was supported by Italian Ministry of University Grants FIRB 2003 and PRIN 2005. The 800-MHz spectrometer was funded by the European Union (Fonds EuropéendeDéveloppement Régional), French Research Ministry, Region Nord-Pas de Calais, University of Sciences and Technology of Lille, and CNRS. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed E-mail: trost{at}alma.unibo.it.

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