Analysis of the High-Order Conformational Changes in Glyceraldehyde-3-phosphate Dehydrogenase Induced by Nicotinamide Adenine Dinucleotide, Adenosine Triphosphate, and Oxidants

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in glycolysis. Beyond this normal function, GAPDH acts as a moonlighting protein, interacting with nonglycolytic molecules to fulfill additional roles, such as apoptosis induction. However, the three-dimensional (3D) structural details underlying these interactions remain unclear, likely due to their dynamic and transient nature. To address this issue, we investigated the structural properties of human and porcine GAPDH using a combination of biophysical techniques, including nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, gel filtration chromatography, and thermal shift assays, with a particular focus on their 3D structures. Our results revealed that although GAPDH becomes unstable upon nicotinamide adenine dinucleotide (NAD⁺) depletion (apo state), its oligomeric structure as a tetramer remains preserved regardless of temperature. In contrast, the presence of adenosine triphosphate (ATP) promotes dimerization at low temperatures, as previously reported. Furthermore, our NMR data suggest that ATP binding exposes the dimer interface and increases the flexibility of side chains in this region. These findings indicate that GAPDH maintains a stable tetrameric structure in the presence of NAD⁺ but becomes structurally unstable and likely more susceptible to oxidation upon NAD⁺ depletion. Additionally, our analyses showed that partial nitrosylation of GAPDH subunits does not induce significant tertiary structural changes. However, significant structural alterations were observed when all four subunits were nitrosylated, although the possibility remains that residues other than the active site residue, Cys152, may have been oxidized. We propose that NAD⁺ depletion, along with oxidation or nitrosylation─most likely at Cys152─destabilizes the GAPDH conformation, and that subsequent ATP binding promotes dimerization. This subunit dissociation may serve as a structural basis for GAPDH’s interactions with other molecules and its moonlighting functions.