Analysis of the high-order conformational changes in glyceraldehyde-3-phosphate dehydrogenase induced by nicotinamide adenine dinucleotide, adenosine triphosphate, and oxidants

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the sixth step of glycolysis. As a moonlighting protein, GAPDH interacts with other molecules, seemingly unrelated to glycolysis, to exert additional functions, such as inducing apoptosis. However, specific mechanisms underlying these interactions remain unclear. Therefore, in this study, we aimed to elucidate these mechanisms by analyzing human and porcine GAPDHs based on their three-dimensional (3D) structures using various biophysical techniques, including nuclear magnetic resonance spectroscopy, gel filtration chromatography, and thermal shift assay. Although GAPDH became unstable when nicotinamide adenine dinucleotide (NAD+) was removed (apo state), the 3D structure of the tetramer was maintained regardless of the temperature. However, in the presence of adenosine triphosphate (ATP), GAPDH split into dimers at low temperatures, exposing the interface between the dimers and increasing the flexibility of the side chains at the site. Moreover, subunit splitting also occurred upon exposure to oxidizing agents, such as hydrogen peroxide. These findings suggest that GAPDH maintains a stable tetramer in the presence of NAD+ but becomes unstable and easily oxidized upon NAD+ depletion. When multiple residues, including those other than the cysteine residue at the active site, are oxidized by reactive oxygen species or nitric oxide, or when it interacts with ATP, GAPDH splits into dimers. This subunit splitting may trigger interactions with other molecules.