Mapping of the interaction site of CP12 with glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii

Lebreton, Sandrine and Andreescu, Simona and Graciet, Emanuelle and Gontero, Brigitte (2006) Mapping of the interaction site of CP12 with glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii. FEBS Journal, 273 (14). pp. 3358-3369. ISSN 1742-464X

Download (262kB) | Preview

Share your research

Twitter Facebook LinkedIn GooglePlus Email more...

Add this article to your Mendeley library


The 8.5 kDa chloroplast protein CP12 is essential for assembly of the phosphoribulokinase ⁄ glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complex from Chlamydomonas reinhardtii. After reduction of this complex with thioredoxin, phosphoribulokinase is released but CP12 remains tightly associated with GAPDH and downregulates its NADPH-dependent activity. We show that only incubation with reduced thioredoxin and the GAPDH substrate 1,3-bisphosphoglycerate leads to dissociation of the GAPDH⁄ CP12 complex. Consequently, a significant twofold increase in the NADPHdependent activity of GAPDH was observed. 1,3-Bisphosphoglycerate or reduced thioredoxin alone weaken the association, causing a smaller increase in GAPDH activity. CP12 thus behaves as a negative regulator of GAPDH activity. A mutant lacking the C-terminal disulfide bridge is unable to interact with GAPDH, whereas absence of the N-terminal disulfide bridge does not prevent the association with GAPDH. Trypsin-protection experiments indicated that GAPDH may be also bound to the central a-helix of CP12 which includes residues at position 36 (D) and 39 (E). Mutants of CP12 (D36A, E39A and E39K) but not D36K, reconstituted the GAPDH⁄ CP12 complex. Although the dissociation constants measured by surface plasmon resonance were 2.5–75-fold higher with these mutants than with wild-type CP12 and GAPDH, they remained low. For the D36K mutation, we calculated a 7 kcalÆmol)1 destabilizing effect, which may correspond to loss of the stabilizing effect of an ionic bond for the interaction between GAPDH and CP12. It thus suggests that electrostatic forces are responsible for the interaction between GAPDH and CP12.

Item Type: Article
Keywords: CP12; GAPDH; interaction site; intrinsically unstructured protein; protein–protein interactions;
Academic Unit: Faculty of Science and Engineering > Biology
Item ID: 7429
Identification Number: 10.1111/j.1742-4658.2006.05342.x
Depositing User: Emanuelle Graciet
Date Deposited: 02 Sep 2016 08:29
Journal or Publication Title: FEBS Journal
Publisher: Wiley
Refereed: Yes

Repository Staff Only(login required)

View Item Item control page

Document Downloads

More statistics for this item...