The small protein CP12: a protein linker for supramolecular complex assembly.
CP12 is an 8.5-kDa nuclear-encoded chloroplast protein, isolated from higher plants. It forms part of a core complex of two dimers of phosphoribulokinase (PRK), two tetramers of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and CP12. The role of CP12 in this complex assembly has not been determined. To address this question, we cloned a cDNA encoding the mature CP12 from the green alga Chlamydomonas reinhardtii and expressed it in Escherichia coli. Sequence alignments show that it is very similar to other CP12s, with four conserved cysteine residues forming two disulfide bridges in the oxidized CP12. On the basis of reconstitution assays and surface plasmon resonance binding studies, we show that oxidized, but not reduced, CP12 acts as a linker in the assembly of the complex, and we propose a model in which CP12 associates with GAPDH, causing its conformation to change. This GAPDH/CP12 complex binds PRK to form a half-complex (one unit). This unit probably dimerizes due partially to interactions between the enzymes of each unit. Reduced CP12 being unable to reconstitute the complex, we studied the structures of oxidized and reduced CP12 by NMR and circular dichroism to determine whether reduction induced structural transitions. Oxidized CP12 is mainly composed of alpha helix and coil segments, and is extremely flexible, while reduced CP12 is mainly unstructured. Remarkably, CP12 has similar physicochemical properties to those of "intrinsically unstructured proteins" that are also involved in regulating macromolecular complexes, or in their assembly. CP12s are thus one of the few protein families of intrinsically unstructured proteins specific to plants.
Graciet
Emmanuelle
E
Laboratoire d'ingéniérie des protéines et contrôle métabolique, Département Biologie des génomes, Institut Jacques Monod, UMR 7592 CNRS, Universités Paris VI -VII, 2 place Jussieu, 75251 Paris Cedex 05, France.
Gans
Pierre
P
Wedel
Norbert
N
Lebreton
Sandrine
S
Camadro
Jean-Michel
JM
Gontero
Brigitte
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000737:chemistry*; D002942:Circular Dichroism; D017931:DNA Primers; D004220:Disulfides / Q000737:chemistry; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
D017931:DNA Primers; D004220:Disulfides; D010940:Plant Proteins; D011994:Recombinant Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase