Construction of a 3D model of CP12, a protein linker

https://doi.org/10.1016/j.jmgm.2005.12.003Get rights and content

Abstract

The chloroplast protein CP12 is known to play a leading role in a complex formation with the enzymes GAPDH and PRK. As a preliminary step towards the understanding of the complex formation mechanism and the exact role of this protein linker, a comparative modelling of the CP12 protein of the green alga Chlamydomonas reinhardtii was performed. Because of the very few structural information and poor template similarities, the derivation of the model consisted in an iterative trial-and-error procedure using the comparative modelling program MODELLER, the following three structure validation programs PROCHECK, PROSA, and WHATIF, and molecular mechanics energy refinement of the model using the program CHARMM. The analysis of the final model reveals a scaffold of key residues that is believed to be essential in the folding mechanism and that coincides with the residues conserved throughout the CP12 family. Our results suggest that this protein is a typical disordered protein. Finally, the various mechanisms by which the CP12 protein can self-interact or binds to other enzymes are discussed in light of its modelled structure and characteristics.

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 α-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.

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