High Resolution Crystal Structures of the Photoreceptor Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) with Three and Four-bound NAD Molecules.

Aug. 30, 2014

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the oxidative phosphorylation of D-glyceraldehyde 3-phosphate (G3P) into 1,3-diphosphoglycerate (BGP) in the presence of the NAD cofactor. GAPDH is an important drug target because of its central role in glycolysis, and non-glycolytic processes such as nuclear RNA transport, DNA replication/repair, membrane fusion and cellular apoptosis. Recent studies found that GAPDH participates in the development of diabetic retinopathy and its progression after the cessation of hyperglycemia. Here, we report two structures for native bovine photoreceptor GAPDH as a homotetramer with differing occupancy by NAD, bGAPDH(NAD)4 and bGAPDH(NAD)3 . The bGAPDH(NAD)4 was solved at 1.52 Å, the highest resolution for GAPDH. Structural comparison of the bGAPDH(NAD)4 and bGAPDH(NAD)3 models revealed novel details of conformational changes induced by cofactor binding, including a loop region (residues 54 - 56). Structure analysis of bGAPDH confirmed the importance of Phe34 in NAD binding, and demonstrated that Phe34 was stabilized in the presence of NAD but displayed greater mobility in its absence. The oxidative state of the active site Cys149 residue is regulated by NAD binding, because this residue was found oxidized in the absence of dinucleotide. The distance between Cys149 and His176 decreased upon NAD binding and Cys149 remained in a reduced state when NAD was bound. These findings provide an important structural step for understanding the mechanism of GAPDH activity in vision and its pathological role in retinopathies.

Figure 1. Characterization of bovine GAPDH. A. SDS-PAGE analysis of bGAPDH purified from bovine ROS. B. Size exclusion profile of purified bGAPDH. The column (Superdex 200, 10/300 GL, GE Healthcare) was calibrated with protein standards (Insert). C. Enzyme activity. Purified bGAPDH (0.5 µg/100 µL) was incubated with 0.2 mM NAD and increasing concentrations of G3P (top panel), or with 1 mM G3P and increasing concentrations of NAD (middle panel). After 30 min of incubation, GAPDH activity was monitored by measuring NADH production at 340 nm. Kinetic measurements were then done over a 45 min time course in the presence of 1 mM NAD and 1 mM G3P (bottom panel).

Results from: Baker BY, Shi W, Wang B, Palczewski K. Protein Sci. 2014 Aug 30. doi: 10.1002/pro.2543. [Epub ahead of print]