Abstract

Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) is a highly abundant enzyme in cells, performing numerous functions in various oligomeric forms. The stable cytoplasmic homo-tetramer plays the primary (glycolytic) role. Here, the focus is the unstable, nuclear monomer (subunit), implicated in DNA replication, repair, and packing into chromosomes. Insight into enzyme function, via temporal sampling of conformational changes and transitions
between binding states, is difficult to achieve using experiment. Functional loop (S-Loop) motion and cofactor (NAD+) unbinding of the Human Placental subunit structure (PDB Entry 1U8F) were studied using classical, all-atom Molecular Dynamics (MD) simulations. Conformational trajectories, generated using conventional MD simulations, revealed that S-Loop motion consists of pivoting (mainly) and internal deformation, and that this motion becomes more concerted (increased magnitude and directionality) when NAD+ is bound to the subunit. Ligand unbinding trajectories, generated using steered and umbrella sampling MD simulations, revealed that NAD+ unbinding is unfavourable, but still feasible if the adenine ribose is pulled away from the subunit while its S-Loop is pivoted away from the NAD+ domain, and entails among other things, Asp-35 unbinding, as well as Lys-194 binding and subsequent unbinding. The present work reinforces the vital role of bound NAD+ in bringing order to subunit S-Loop motion, which in turn is essential for maintaining inter-subunit interactions in the GAPDH tetramer.