Investigating the functional dynamics of glyceraldehyde-3-phosphate dehydrogenase upon ligand binding at the putative allosteric binding sites

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an attractive target to combat infection-related diseases as antibiotic resistance poses a global threat. Here, we investigated the functional dynamics of methicillin-resistant S. aureus GAPDH (SaGAPDH) in apo-form and with ligands bound at two distinct potential allosteric binding sites in its tunnel-like region. AutoDock Vina was used for flexible docking with a library of 2447 FDA-approved drugs. After the interaction analysis and chemical fragment clustering, 5 compounds mutual to both sites were selected and subjected to independent 3 × 500 ns-long molecular dynamics (MD) simulations coupled with Molecular Mechanics Generalized Born Surface Area calculations to estimate their binding free energies. The ligand-protein dynamics pointed to either an increase or a decrease in the solvent accessibility of the co-factor NAD⁺ binding site as compared to apo-dynamics without an apparent change in residue fluctuations. Furthermore, dihedral angles of the co-factor binding site residues, particularly R12 and N316 changed upon ligand binding to the tunnel-like region. Residue network models based on the MD trajectories for each system revealed potential allosteric communication pathways linking putative allosteric binding sites to the co-factor binding sites. Favorable interactions between the ligands and the previously suggested hub residues T49-R53, E204 (S-loop) and/or Y180 seemed to affect the solvent accessibility of the binding sites that can either facilitate or prevent NAD⁺ binding. Dynamic coupling of NAD⁺ binding sites through correlated residue fluctuations was consistent in all investigated systems, revealing how cooperativity between the functional sites can be maintained by SaGAPDH. All findings suggested hit compounds and the putative allosteric binding sites to change the NAD⁺ binding site dynamics that should be further evaluated by in vitro studies to assess their antibacterial activities.