A Trajectory of Discovery: Metabolic Regulation by the Conditionally Disordered Chloroplast Protein, CP12

Abstract

The chloroplast protein CP12, which is widespread in photosynthetic organisms, belongs to the intrinsically disordered proteins family. This small protein (80 amino acid residues long) presents a bias in its composition; it is enriched in charged amino acids, has a small number of hydrophobic residues, and has a high proportion of disorder-promoting residues. More precisely, CP12 is a conditionally disordered proteins (CDP) dependent upon the redox state of its four cysteine residues. During the day, reducing conditions prevail in the chloroplast, and CP12 is fully disordered. Under oxidizing conditions (night), its cysteine residues form two disulfide bridges that confer some stability to some structural elements. Like many CDPs, CP12 plays key roles, and its redox-dependent conditional disorder is important for the main function of CP12: the dark/light regulation of the Calvin-Benson-Bassham (CBB) cycle responsible for CO₂ assimilation. Oxidized CP12 binds to glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase and thereby inhibits their activity. However, recent studies reveal that CP12 may have other functions beyond the CBB cycle regulation. In this review, we report the discovery of this protein, its features as a disordered protein, and the many functions this small protein can have.

Keywords: Calvin-Benson-Bassham cycle; conditionally disordered protein; history of modern science; metabolism regulation; moonlighting protein; protein-protein interaction.

Figure 1

Schematic representation of the predicted secondary structure of mature CP12. Cysteine residues proposed to form peptide loops and that can form two disulfide bridges (C23–C31 and C66–C75) when CP12 is oxidized are indicated with black circles. These two loops are separated by an alpha helix. The proline residue conserved in CP12 from Plantae is shown with a yellow circle. Numbering is from the C. reinhardtii mature CP12 sequence. This figure was created with BioRender (https://biorender.com/ (accessed on 25 July 2022)) and adapted from Wedel et al. [16].

Figure 2

CP12 behaves as an IDP. (A) SDS-PAGE (12%) of 4 µg C. reinhardtii recombinant CP12 under its oxidized or reduced state. (B) MALDI-ToF mass spectrum of the native CP12 isolated from the PRK/GAPDH/CP12 complex of C. reinhardtii. (C) Size-exclusion chromatography profile of C. reinhardtii recombinant CP12 under oxidized (black) or reduced (red) conditions (column: Superdex 200 10 × 300 mm). Above the chromatogram, the dots from A to G indicate the position of molecular-weight standard globular proteins. A: Ferritine (MW 440 kDa, r_H 6.8 nm); B: Catalase (MW 240 kDa, r_H 5.5 nm); C: dimer of Bovine Serum Albumin (BSA, MW 136 kDa, r_H 4.5 nm); D: monomer of BSA (MW 68 kDa, r_H 3.5 nm); E: Ovalbumin (MW 43 kDa, r_H 3 nm); F: Cytochrome (MW 12.5 kDa, r_H 2 nm) C; and G: oxidized form of DTT. MW and r_H stand for molecular weight and hydrodynamic radius.

Figure 3

CP12 presents a bias in amino acid composition. Comparison of amino acid composition between globular proteins and C. reinhardtii CP12 using composition profiler (http://www.cprofiler.org (accessed on 25 July 2022)) [40]. The globular proteins dataset is from protein data bank (PDB) Select 25.

Figure 4

Biophysical analysis of CP12 confirmed that CP12 is an IDP. (A) Circular dichroism spectra of 10 µM recombinant C. reinhardtii oxidized CP12 (black), and of a CP12 mutant lacking the N-terminal disulfide bridge (mimicking reducing conditions, red). (B) Normalised Kratky representation of the SAXS data of the oxidized (black) and reduced (red) form of recombinant C. reinhardtii CP12. (C) NMR ¹H-¹⁵N-HSQC spectra of the oxidized (black) and reduced (red) form of recombinant C. reinhardtii CP12. The box between 9.5 and 10 ppm corresponds to the insert shown on the left.

Figure 5

Schematic representation of structural variants of CP12. CBS (orange rectangles) stands for cystathionine β-synthase. The pairs of black lines represent cysteine residues pairs, the red rectangles represent the core sequences AWD_VEEL, and the yellow lines represent the central proline within the C-terminal residues pair. Adapted from D.N Stanley et al., 2013 [58].

Figure 6

Atlas of CP12 functions. The core sequence of CP12, AWD_VEEL, is in red, and sulfur atoms are indicated in yellow. Reduced CP12 is fully disordered, and oxidized CP12 is partially ordered. Under oxidized state, CP12 forms a supramolecular complex with GAPDH and PRK. A non-exhaustive list of CP12 functions is indicated in the scheme.