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Creating Order out of Disorder: Structural Imprint of GAPDH on CP12

2011; Elsevier BV; Volume: 19; Issue: 12 Linguagem: Inglês

10.1016/j.str.2011.11.004

1878-4186

Brigitte Gontero, Luisana Avilán,

Mass Spectrometry Techniques and Applications

The work presented by Matsumura et al. in this issue of Structure describes the structure of the ternary GAPDH-NAD-CP12 and the binary NAD-GAPDH complex in the cyanobacterium Synechococcus elongatus. The work presented by Matsumura et al. in this issue of Structure describes the structure of the ternary GAPDH-NAD-CP12 and the binary NAD-GAPDH complex in the cyanobacterium Synechococcus elongatus. CP12 is a chloroplast protein made up of 80 amino acid residues that can be found widely in many photosynthetic organisms including higher plants, microalgae, and cyanobacteria (Groben et al., 2010Groben R. Kaloudas D. Raines C.A. Offmann B. Maberly S.C. Gontero B. Photosynth. Res. 2010; 103: 183-194Crossref PubMed Scopus (51) Google Scholar). Some regions of CP12 are highly conserved, and in most organisms, CP12 has a pair of cysteine residues at the C terminus and/or a second pair at the N terminus, though this second pair is absent in the cyanobacterium, Synechococcus elongatus. Each of these pairs is capable of forming a disulphide bridge. Although the disulphide bridges are likely to structure the molecule, CP12 shares some physico-chemical properties with intrinsically disordered proteins (IDPs). CP12 from the green alga Chlamydomonas reinhardtii and from the higher plant Arabidopsis thaliana were shown, by circular dichroism and nuclear magnetic resonance, to lack any regular secondary structure in solution. Under oxidizing conditions, owing to the formation of two disulphide bridges at the C terminus and at the N terminus of the CP12, the overall disorder of the protein is reduced and the amount of α helices is increased, but oxidized CP12 still remains very flexible. IDPs, mainly found in eukaryotes, exhibit little secondary structure, high flexibility, and low compactness. As a consequence of their plasticity, IDPs bind to multiple partners and, under physiological conditions and the absence of a rigid globular structure, might confer a considerable functional advantage (Tompa, 2005Tompa P. FEBS Lett. 2005; 579: 3346-3354Abstract Full Text Full Text PDF PubMed Scopus (593) Google Scholar). The function of IDPs includes the regulation of translation and transcription, protein phosphorylation, storage of small molecules, signal transduction, and acting as linkers to form multi-protein complexes. It has been shown that CP12 may act as a linker between phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), two enzymes that belong to the Calvin cycle responsible for CO2 assimilation (Graciet et al., 2003Graciet E. Gans P. Wedel N. Lebreton S. Camadro J.M. Gontero B. Biochemistry. 2003; 42: 8163-8170Crossref PubMed Scopus (98) Google Scholar). It has also been reported that CP12 is able to bind copper ion and FBP aldolase, another Calvin cycle enzyme (Erales et al., 2008Erales J. Avilan L. Lebreton S. Gontero B. FEBS J. 2008; 275: 1248-1259Crossref PubMed Scopus (36) Google Scholar, Erales et al., 2009Erales J. Gontero B. Whitelegge J. Halgand F. Biochem. J. 2009; 419: 75-82Crossref PubMed Scopus (32) Google Scholar). The active role of CP12 in the assembly pathway of the PRK/GAPDH/CP12 complex has been extensively described (Figure 1) in C. reinhardtii and in the higher plant, Arabidopsis thaliana (Marri et al., 2008Marri L. Trost P. Trivelli X. Gonnelli L. Pupillo P. Sparla F. J. Biol. Chem. 2008; 283: 1831-1838Crossref PubMed Scopus (60) Google Scholar). CP12 promotes the oligomerisation between PRK and GAPDH in the presence of NAD(H) and their dissociation in the presence of NADP(H). The inactive complex exists under dark or oxidizing conditions when the Calvin cycle is not functioning and dissociates upon light or reducing conditions to release fully-active enzymes. The Calvin cycle may thus be regulated by association and dissociation of the PRK/GAPDH/CP12 complex. There are a number of different isoforms of GAPDH: a GapA and a GapC1 isoform that do not have regulatory cysteine residues, and GapB isoform, mainly found in higher plants (Trost et al., 2006Trost P. Fermani S. Marri L. Zaffagnini M. Falini G. Scagliarini S. Pupillo P. Sparla F. Photosynth. Res. 2006; 89: 1-13Crossref Scopus (75) Google Scholar) which has a C terminus extension that is homologous with part of CP12. The existence of the GAPDH/CP12 complex allows isoform A4 of GAPDH in algae and cyanobacteria to be redox-regulated. The presence of the C terminal disulphide bridge on CP12 offsets the lack of redox sensitive cysteine residues on the A subunit and consequently plays a major role in the regulation of the activity of the A4 GAPDH isoform. Studies using mutagenesis and limited proteolysis have allowed the residues involved in the interaction between CP12 and GAPDH from C. reinhardtii to be mapped. A bioinformatic approach has also been used to produce a structural model of oxidized CP12 from the C. reinhardtii sequence, but no experimental data were available (Gardebien et al., 2006Gardebien F. Thangudu R.R. Gontero B. Offmann B. J. Mol. Graph. Model. 2006; 25: 186-195Crossref PubMed Scopus (38) Google Scholar). The work presented in this issue of Structure by Matsumura et al., 2011Matsumura H. Kai A. Maeda T. Tamoi M. Satoh A. Tamura H. Hirose M. Ogawa T. Kizu N. Wadano A. et al.Structure. 2011; 19 (this issue): 1846-1854Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar provides structural information required to understand the functional biology of GAPDH regulation, at least for cyanobacteria. The crystal of GAPDH-NAD-CP12 confirmed that oxidized CP12 interacts within the GAPDH groove, and the structure of this ternary complex explains how CP12 binding downregulates the activity of GAPDH. IDPs may undergo some degree of folding upon binding to their partner, and clearly, the work by Matsumura et al., 2011Matsumura H. Kai A. Maeda T. Tamoi M. Satoh A. Tamura H. Hirose M. Ogawa T. Kizu N. Wadano A. et al.Structure. 2011; 19 (this issue): 1846-1854Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar showed that CP12 became partially structured when bound to GAPDH. Few studies have analyzed the structure of protein-protein complexes in the Calvin cycle. To our knowledge, the only available crystal structure of a complex involved in this pathway was the structure of a three-enzyme complex (phosphoribose isomerase/PRK/ribulose 1,5-bisphosphate carboxylase-oxygenase) from spinach obtained at 3.5 Å resolution (Sainis and Jawali, 1994Sainis J.K. Jawali N. Indian J. Biochem. Biophys. 1994; 31: 215-220PubMed Google Scholar). Using cryo-electron microscopy for the C. reinhardtii PRK/GAPDH/CP12 complex, strong structural differences between the modeled PRK dimers (not embedded in the complex) and PRK in the three-dimensional reconstruction volume of the whole complex were suggested (Mouche et al., 2002Mouche F. Gontero B. Callebaut I. Mornon J.P. Boisset N. J. Biol. Chem. 2002; 277: 6743-6749Crossref PubMed Scopus (25) Google Scholar). Most isolated particles had a rod-like shape, with overall dimensions (20 × 10 nm) that were in good agreement with the expected size of the complex (about 460 kDa). At that time, the authors had not detected CP12 in the bi-enzyme complex, but the data clearly showed the presence of additional density that was postulated to be CP12. These data were a first step for a better understanding of conformational changes within multi-enzyme complexes but the mechanistic details were lacking due to low resolution. Together with the studies performed earlier, the crystal structure of the GAPDH-NAD-CP12 ternary complex obtained by Matsumura et al., 2011Matsumura H. Kai A. Maeda T. Tamoi M. Satoh A. Tamura H. Hirose M. Ogawa T. Kizu N. Wadano A. et al.Structure. 2011; 19 (this issue): 1846-1854Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar from S. elongatus allows a better understanding of the regulation of GAPDH with CP12 in cyanobacteria and also raises some important questions that can now be addressed. For example, does PRK binding depend on extensive negative charge that emerged upon CP12 binding on GAPDH? Further, work is now required to see if CP12 becomes fully structured in the presence of GAPDH and PRK. Moreover, because the CP12 from the cyanobacterium S. elongatus does not possess the N-terminal disulphide bridge, a comparison with other photosynthetic organisms that possess a CP12 with two disulphide bridges would be very interesting. It is clear that further analysis of this structure will provide more insights into the precise molecular interactions, at the atomic level, underlying enzyme regulation. Structure Basis for the Regulation of Glyceraldehyde-3-Phosphate Dehydrogenase Activity via the Intrinsically Disordered Protein CP12Matsumura et al.StructureDecember 07, 2011In BriefThe reversible formation of a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-CP12-phosphoribulokinase (PRK) supramolecular complex, identified in oxygenic photosynthetic organisms, provides light-dependent Calvin cycle regulation in a coordinated manner. An intrinsically disordered protein (IDP) CP12 acts as a linker to sequentially bind GAPDH and PRK to downregulate both enzymes. Here, we report the crystal structures of the ternary GAPDH-CP12-NAD and binary GAPDH-NAD complexes from Synechococcus elongates. Full-Text PDF Open Archive