Construction of a 3D model of CP12, a protein linker
Introduction
CP12 is a small nuclear-encoded 8.5 kDa chloroplast protein. This protein is also found across photosynthetic prokaryotic systems in cyanobacteria. For the green alga Chlamydomonas reinhardtii, it was shown that chloroplastic CP12 in its oxidized form acts as a protein linker between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), thus providing evidence for a novel way for the light regulation of photosynthetic enzymes in the Calvin cycle pathway [1], [2]. A model for the binding pathway between CP12 and its target enzymes has been experimentally derived [1], [3]. Nevertheless, the structural basis for the mechanism underlying this interaction has yet to be demonstrated though low resolution of the whole complex was studied using cryoelectron microscopy [4]. The presence of CP12 in its oxidized form appears to be crucial in the complex formation. It should furthermore be pointed out that the CP12 sequence of this particular alga is very similar to the sequences reported for many prokaryotic and eukaryotic species.
To better grasp the role of CP12 in the complexation mechanism between GAPDH, PRK, and CP12, knowledge of the tertiary structure of this protein is essential. No experimental structure of any CP12 homologues has been reported so far and NOE experiments conducted for this protein in C. reinhardtii remain difficult to interpret [1]. However, experimental results clearly indicate the presence of two disulphide bridges when CP12 is in its oxidized state, one between the two N-ter cysteines and one between the two C-ter cysteines. These cysteines are highly conserved among prokaryotic and eukaryotic CP12 accessions. As reported previously, the formation of these disulphide bridges upon oxidation plays a key role in the folding of CP12 [1]. Information available from NMR and circular dichroism showed that oxidized CP12 is mainly composed of -helices and coil segments and is very flexible, while reduced CP12 is mainly unstructured [1].
In this study, our aim was to derive a model of the tertiary structure of the oxidized form of the CP12 protein found in C. reinhardtii by using a comparative modelling approach. A drawback arises, however, from the fact that no homologues was found for this 80 residues long mature peptide despite extensive investigation. To overcome this problem, our strategy was to consider an extensive iterative trial-and-error procedure that combines the following three steps: generation of models by comparative modelling, validation of a model by ad hoc structure validation programs, and energy refinement using molecular mechanics calculations.
The paper is organized as follows. In Section 2, all the details about the procedure we followed to derive the models are given. In Section 3, we first present the results of the comparative modelling calculation (Sections 3.1 Comparative modelling, 3.2 Analysis of the model). Then, we discuss the mechanisms by which CP12 self-interacts (complexation can be observed) or binds to the enzymes GAPDH and PRK in light of its structure and characteristics (Section 3.3). Finally, a comparative discussion between the tertiary structure here reported and other disordered proteins is proposed (Section 3.4).
Section snippets
Selection of templates and input preparation
Prediction of the secondary structure for the 80 residues long mature peptide of CP12 from C. reinhardtii was performed through the JPRED server (http://www.compbio.dundee.ac.uk/www-jpred) [5]. The results indicate two long helices between residues 8 and 23 and between 29 and 51; a shorter helix is also predicted between residues 61 and 65. Besides, as derived experimentally [2], all thiol groups from the four cysteines are expected to be in the oxidized form, thereby resulting in two S–S bonds
Comparative modelling
The modelling approach can be summarized as follows. From an initial alignment, it consists in four steps: (i) the derivation of sets of models by MODELLER, (ii) the validation tests (PROSA and WHATIF) made for a few models that are chosen from their relative objective function values, (iii) an energy minimization run is subsequently considered for the structures that have passed these tests, and (iv) a final validation step (as in (ii)). The alignment was manually modified and process
Conclusion
In the process of modelling CP12, we had to face two major concerns. In first instance, despite the absence of homologous structures from structural databases, we were able to identify useful templates that share low sequence similarity with CP12 which, when combined together, encompass the whole length of CP12. In second instance, we were able to derive an additional set of useful constraints from both predictive methods and experimental data thereby overcoming the fact that critical mass is
Acknowledgements
R. Thangudu is currently supported by a PhD grant from the Conseil Régional de La Réunion. The authors are most thankful to Dr. R. Sowdhamini for fruitful discussions.
References (29)
- et al.
Striking conformational change suspected within the phosphoribulokinase dimer induced by interaction with gapdh
J. Biol. Chem.
(2002) - et al.
Comparative protein modelling by satisfaction of spatial restraints
J. Mol. Biol.
(1993) What if: a molecular modelling and drug design program
J. Mol. Graph.
(1990)- et al.
Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes
J. Comput. Phys.
(1977) - et al.
Thioredoxin activation of phosphoribulokinase in a bi-enzyme complex from Chlamydomonas reinhardtii chloroplasts
J. Biol. Chem.
(2000) - et al.
Analysis of the structural properties of creb and phosphorylated creb
J. Biol. Chem.
(1996) - et al.
Solution structure of the kix binding domain of cbp bound to the transactivation domain of creb: a model for activator:coactivator interactions
Cell
(1997) - et al.
Protein disorder prediction. implications for structural proteomics
Structure (Camb)
(2003) - et al.
Prediction and functional analysis of native disorder in proteins from the three kingdoms of life
J. Mol. Biol.
(2004) - et al.
The structure of the -catenin/E-cadherin complex and the molecular basis of diverse ligand recognition by -catenin
Cell
(2001)
Crystal structure of the flagellar sigma/anti-sigma complex sigma(28)/flgm reveals an intact sigma factor in an inactive conformation
Mol. Cell
The small protein CP12: a protein linker for supramolecular complex assembly
Biochemistry
Cp12 provides a new mode of light regulation of Calvin Cycle activity in higher plants
Proc. Natl. Acad. Sci. USA
Emergence of new regulatory mechanisms in the benson-calvin pathway via protein–protein interactions: a glyceraldehyde-3-phosphate dehydrogenase/CP12/phosphoribulokinase complex
J. Exp. Bot.
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2015, Biochemical and Biophysical Research CommunicationsCitation Excerpt :In vivo, the inherent flexibility and structural adaptability of CP12 makes it an adapter between the tetrameric GAPDH and the dimeric PRK in Calvin cycle. A 3D model of CP12 based on bioinformatics approach shows the four cysteine residues and all lysine positions (Fig. 1A) [26]. In vitro reconstitution assays showed that CP12 mutants were unable to form the dimeric unit (GAPDH–CP12–PRK)2 complex (Fig. 1B).