The crystal structure of photosynthetic A2B2-glyceraldehyde-3-phosphate dehydrogenase discloses the mechanism of thioredoxin regulation

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a light-regulated, NAD(P)H-dependent enzyme involved in plant photosynthetic carbon reduction. At difference with lower photosynthetic organisms which only contain A4-GAPDH, the major GAPDH isoform of land plants is made up of A and B subunits, the latter containing a C-terminal extension (CTE) with fundamental regulatory functions. Light-activation of AB-GAPDH depends on the redox state of a pair of CTE cysteines forming a disulfide bond under control of thioredoxin f, leading to specific inhibition of the NADPH-dependent activity. The crystallographic structural model of oxidized A2B2-GAPDH complexed with NADP is composed of one tetramer and a dimer, which generates a second tetramer using a crystallographic two-fold axis coincident with the molecular axis P. A2B2- GAPDH showed an overall structural organization similar to thioredoxin-independent A4-GAPDH [2]. These two isoforms almost identical along P and Q axes, are differently sized along R axis, where the A2B2 tetramer (75.7 \uc5) appeared shorter than A4-GAPDH (78.1 \uc5). Each A or B subunit consists of a coenzyme-binding domain and a catalytic domain. One NADP molecule was bound to each coenzyme domain, and one or two sulphate ions were found in each catalytic domain. The CTE of B-subunits was only partially detectable by x-ray diffraction experiments. The inspection of the electron density maps showed an elongated electron density region not continuous with any subunit, but interpretable as a protein chain slipping into the cleft bordered by a pair of A and B-subunits. This cleft features a positive electrostatic surface potential due to six exposed arginines and two histidines (Arg77, Arg183, Arg191 and His190), conserved in both A and B-subunits. The CTE contains negatively charged residues and could be attracted by the strongly cationic cleft. Present data support the view that A2B2-GAPDH inhibition by thioredoxin depends on the docking of the oxidized CTE into the cleft delimited by A/B-subunits. In this location, the CTE appears to interfere with the recognition of bound NADP by the crucial residues Arg77 and Ser188, thus leaving the tetramer in a kinetically inhibited conformation, unable to efficiently use NADPH as the preferred coenzyme