Introduction: Cancer is now recognized as the leading cause of death in many countries. One of the hallmarks of cancer is the reprogramming of cellular metabolism. This means that because cancer cells require a lot of nutrients to support their rapid growth and proliferation, the metabolism in these cells is altered compared to normal cells. These changes include increased aerobic glycolysis (the conversion of glucose to lactate even in the presence of oxygen), increased activity of the pentose phosphate pathway (PPP), and decreased oxidative phosphorylation. The PPP is a branch of glycolysis that produces ribose-5-phosphate (a precursor for nucleotide synthesis) and NADPH, which contribute significantly to the production of building blocks required for cancer cell growth. Glucose-6-phosphate dehydrogenase (G6PD), the first and rate-limiting enzyme in this pathway, is overexpressed and overactivated in many cancers. The dimer and tetramer forms of this enzyme are active, while the monomer form is inactive. Since protein-protein interactions between G6PD subunits are essential for its activity, preventing its assembly process could be considered a therapeutic strategy.
Methods: In this study, considering the necessity of enzyme subunit assembly for activation, the dimer and tetramer interfaces were investigated using computational methods that could be useful in designing inhibitors of this process. For this purpose, in silico methods, including molecular dynamics simulation, were used to investigate the dynamic properties of the G6PD enzyme in monomer, dimer, and tetramer forms. Two enzyme structures with PDB codes 2BH9 and 2BHL were used for dimer and tetramer studies, respectively. Molecular dynamics simulations were performed for 200 ns using the pmemd module in the Amber20 Linux software package. Before starting the simulation, the inpcrd and prmtop files were prepared in the xleap program by applying the ff19SB force field and creating a truncated octahedral box with the OPC water model 10 Å around the protein. The simulation was performed in three stages of energy minimization, system temperature increase/equilibration, and production. Finally, the simulation trajectory analysis was performed in the cpptraj program, and the key dynamic parameters were plotted and analyzed using the Xmgrace software. In addition, the COCOMAPS web server was used to study the contact map and the distance between residues.
Results: RMSD analysis over 200 ns of molecular dynamics simulations was performed to evaluate the structural stability and equilibration of G6PD in its three oligomeric states. The results indicate that the monomer exhibits larger fluctuations and lower stability compared to the dimer and tetramer. The sharp deviations observed in the monomer at 45 and 117 ns are likely associated with conformational changes. RMSF analysis further reveals higher flexibility in the monomer, particularly in regions involved in dimer interface interactions and in binding the structural NADP within the C-terminal domain. Analysis of the radius of gyration suggests reduced structural compactness of the monomer relative to the other forms. Furthermore, hydrogen bond analysis shows that the number of hydrogen bonds at the dimer interface is substantially higher than that at the tetramer interface. In the following, intermolecular contact maps obtained using a cutoff distance of 8 Å show that the dimer interface has a more extensive contact area compared to the tetramer interface.
Conclusion: Overall, our findings indicate that interactions between G6PD subunits, particularly hydrogen bonds and hydrophobic contacts that stabilize the dimeric and tetrameric assemblies, result in reduced mobility and structural fluctuations, especially at the interface regions. This enhanced stability in the multimeric states is essential for proper enzyme activity.