Molecular Mechanism of NADPH-Glyceraldehyde-3-phosphate Dehydrogenase Regulation through the C-Terminus of CP12 in Chlamydomonas reinhardtii

Abstract

In Chlamydomonas reinhardtii, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) consists of four GapA subunits. This A4 GAPDH is not autonomously regulated, as the regulatory cysteine residues present on GapB subunits are missing in GapA subunits. The regulation of A4 GAPDH is provided by another protein, CP12. To determine the molecular mechanisms of regulation of A4 GAPDH, we mutated three residues (R82, R190, and S195) of GAPDH of C. reinhardtii. Kinetic studies of GAPDH mutants showed the importance of residue R82 in the specificity of GAPDH for NADPH, as previously shown for the spinach enzyme. The cofactor NADPH was not stabilized through the 2'-phosphate by the serine 195 residue of the algal GAPDH, unlike the case in spinach. The mutation of R190 also led to a structural change that was not observed in the spinach enzyme. This mutation led to a loss of activity for NADPH and NADH, indicating the crucial role of this residue in maintaining the algal GAPDH structure. Finally, the interaction between GAPDH mutants and wild-type and mutated CP12 was analyzed by immunoblotting experiments, surface plasmon resonance, and kinetic studies. The results obtained with these approaches highlight the involvement of the last residue of CP12, Asp80, in modulating the activity of GAPDH by preventing access of the cofactor NADPH to the active site. These results help us to bridge the gap between our knowledge of structure and our understanding of functional biology in GAPDH regulation.

Conformational modulation and hydrodynamic radii of CP12 protein and its complexes probed by fluorescence correlation spectroscopy

“…Based on the similar fluorescence brightness found for CP12 mutant proteins alone and for the sub‐complexes, only one monomer of CP12C31S or CP12C75S bound to GAPDH and PRK respectively. The mutant CP12C31S behaves as CP12 locked in a reduced state and therefore this result is in agreement with previous results where only one molecule of reduced CP12 was bound to GAPDH while two molecules of oxidized CP12 were bound to tetrameric GAPDH . The present study also indicates that the ratio 1 : 4 of CP12 to PRK is sufficient to form the CP12C75S–PRK complex; with a K D of 1.6 μ m determined by surface plasmon resonance (SPR) and the experimental conditions used in this report, one would expect about 20% of complex formation between CP12 and PRK.…”

Conformational Selection and Folding-upon-binding of Intrinsically Disordered Protein CP12 Regulate Photosynthetic Enzymes Assembly

“…Few (and variable) interactions stabilize this binding; often one single interaction linking the ␣-helix-C to GAPDH (Arg-191) and an ionic bond between the side chains of Glu-69 (CP12) and Arg-77 (GAPDH). The essential role of these two GAPDH arginines in CP12 binding was proved in the orthologous Chlamydomonas system (51,52) and confirmed by the crystallographic structure of the binary complex from S. elongatus (16). Therefore, the GAPDH-CP12 encounter complex is the result of a preliminary, albeit essential, conformational selection performed by GAPDH on the CP12 structural ensemble.…”

Comparative Analysis of 126 Cyanobacterial Genomes Reveals Evidence of Functional Diversity Among Homologs of the Redox-Regulated CP12 Protein

“…Previous analysis has shown that the C-terminal loop of CP12 is critical for the interaction with GAPDH (Erales et al, 2011;Matsumura et al, 2011;Fermani et al, 2012). Glu-69 is thought to be important in interactions with PRK, GAPDH, and NAD and is particularly well conserved among cyanobacterial CP12 proteins (Matsumura et al, 2011).…”

“…However, a recent study of chloroplast CBS proteins in higher plants suggests that they are involved in the activation of thioredoxins in the ferredoxin-thioredoxin system and that binding AMP in the chloroplast increases this activation, thereby enabling Interacts with phosphate and Rib group Interaction c a GAPDH binding also seen by Fermani et al (2012). b Importance for GAPDH binding also suggesting by Erales et al (2011). c Copper interaction seen by Erales et al (2009a).…”