Structure Basis for the Regulation of Glyceraldehyde-3-Phosphate Dehydrogenase Activity via the Intrinsically Disordered Protein CP12

Abstract

The 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. The GAPDH-CP12 complex structure reveals that the oxidized CP12 becomes partially structured upon GAPDH binding. The C-terminus of CP12 is inserted into the active-site region of GAPDH, resulting in competitive inhibition of GAPDH. This study also provides insight into how the GAPDH-CP12 complex is dissociated by a high NADP(H)/NAD(H) ratio. An unexpected increase in negative charge potential that emerged upon CP12 binding highlights the biological function of CP12 in the sequential assembly of the supramolecular complex.

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

“…Furthermore, the CP12-C group contains a subset of 39 sequences, found only in marine picoplanktonic cyanobacteria, that lack the otherwise conserved core CP12 AWD_VEEL sequence (CP12-C-M; residues 66-73 in CP12-N/C logo). This core sequence is implicated in GAPDH binding by trypsinprotection experiments (Lebreton et al, 2006); however, this region of CP12 is not visible in the electron density of either of the CP12-GAPDH structures (Matsumura et al, 2011;Fermani et al, 2012). Additionally, the N-terminal Gly-24, Ser-27, and His-46 and C-terminal Pro-65 residues are strongly conserved among all CP12 homologs (including those from eukaryotes), while the Gln-41 and Phe-57 residues following the AWD_VEEL sequence that have previously been noted as specific to cyanobacteria (Groben et al, 2010) are not strongly conserved among all cyanobacterial variants ( Fig.…”

“…By a combination of sequence comparison and homology modeling using the coordinates of the S. elongatus CP12-C model from the crystal structure as a template, we evaluated the potential of the different cyanobacterial CP12 types to form a complex with their cognate GAPDH, coordinate copper, and bind NAD (Matsumura et al, 2011;Protein Data Bank [PDB] no. 3B1J).…”

“…Two types of GAPDH are present in cyanobacteria: type 1 contains the residues identified by Matsumura et al (2011) as being important for CP12 binding , and the copper-interacting Cys-155, Thr-156, and His-182). A second type (type 2) of GAPDH is also found in cyanobacteria; this paralog contains the copper-interacting residues but not the other CP12-binding residues.…”

“…Recently, structures have been determined of both S. elongatus and Arabidopsis CP12 in complex with GAPDH (Matsumura et al, 2011;Fermani et al, 2012). These structures provide unprecedented detail of the intermolecular interactions between CP12 and GAPDH and suggest how PRK may be recruited to the complex.…”

“…The C-terminal domain of CP12 (residues 53-75 of S. elongatus CP12) inserts into the active site of GAPDH, while the N terminus of CP12 (residues 1-52) remains disordered in the CP12-GAPDH complex and, presumably, interacts with PRK. In S. elongatus, CP12 binding to GAPDH creates a patch of negative charge on the binary complex that is proposed to electrostatically attract positive charges on PRK (Matsumura et al, 2011). However, this may be a feature specific to cyanobacterial CP12, because this negatively charged patch is not present in the Arabidopsis structure (Fermani et al, 2012).…”