Structural and Functional Impact of the G340S Mutation in Plasmodium falciparum Ferredoxin NADP⁺ Reductase: In silico...

Plasmodium falciparum, the deadliest malaria parasite, continues to burden health systems across sub-Saharan Africa, where artemether-lumefantrine remains a frontline treatment. Partial artemisinin resistance is emerging, increasing selection pressure on lumefantrine (LM), yet the molecular basis of reduced LM susceptibility remains poorly defined. While LM has been described as refractory to resistance, its efficacy could be compromised if resistance arises. To explore candidate determinants of LM response and guided by a Gly332Ser substitution previously identified in Ferredoxin NADP ⁺ reductase from LM-selected Plasmodium berghei, we investigated the homologous G340S variant in Plasmodium falciparum ferredoxin NADP ⁺ reductase ( PfFNR), a central enzyme of the apicoplast redox system. To predict the structural and functional impact of the G340S substitution, this study integrated sequence conservation analysis, AlphaFold 3 structural modelling, structural superimposition, molecular dynamics simulation, NADPH and NADP ⁺ docking, HADDOCK docking with P. falciparum ferredoxin ( PfFd), and comparative docking of lumefantrine, primaquine, artemisinin and dihydroartemisinin. Gly340 was conserved across the Plasmodium FNR orthologs examined and mapped to a constrained loop adjacent to the NADP ⁺ binding region. Structural superimposition showed that G340S preserved the global PfFNR fold but introduced localized changes near residue 340. Exploratory single-trajectory molecular dynamics suggests that G340S may alter PfFNR conformational sampling, with increased RMSD, radius of gyration, solvent-accessible surface area and residue-level flexibility. Cofactor docking predicted a modest mutation-associated shift, strongest for NADPH, whereas NADP ⁺ docking remained largely stable. HADDOCK analysis indicated altered PfFNR-PfFd docking preferences, and antimalarial docking showed ligand-dependent effects, greatest for artemisinin and dihydroartemisinin, with minimal lumefantrine change. Together, these data support G340S as a redox-associated candidate variant that may reshape cofactor and partner-interface, but not as a validated lumefantrine-resistance marker until confirmed by biochemical, genetic and parasite-based assays. The findings define testable mechanisms for future functional assays of PfFNR in antimalarial response phenotypes.