Extended data: Structural and Functional Impact of the G340S Mutation in Plasmodium falciparum Ferredoxin NADP⁺ Reductase: In silico Analysis and Molecular Docking.

Plasmodium falciparum, the deadliest malaria parasite, continues to burden health systems across sub-Saharan Africa. To reduce this burden, Artemisinin-based combination therapies (ACTs), particularly artemether-lumefantrine (AL), remain the frontline treatment. However, partial artemisinin resistance is spreading, exerting selection pressure on lumefantrine (LM), an essential long-acting partner drug. While LM has been described as refractory to resistance, its efficacy could be compromised if resistance arises. To elucidate resistance markers associated with LM, our preliminary studies mapped a mutation in Plasmodium berghei Ferredoxin NADP⁺ Reductase (FNR), G332S, corresponding to G340S in P. falciparum. PfFNR is an essential apicoplast enzyme central to key biosynthetic pathways and drug activation. To explore the structural and functional impact of the G340S substitution, this study employed an in silico bioinformatics approach. High-confidence wild-type and mutant PfFNR models were built using AlphaFold3 and I-TASSER, docking was conducted using AutoDock Vina and AlphaFold3, and molecular dynamics (MD) simulations in GROMACS. Structural visualization and interaction mapping were performed in ChimeraX. Structural analysis indicates that G340S changes the conformation of the NADP⁺-binding loop, disrupts hydrogen-bonding networks in the cofactor-overlapping site, and decreases the cofactor-binding affinity without perturbing the overall fold. Docking revealed conserved NADP⁺ and ferredoxin binding with localized disturbances that could influence electron transfer. MD simulations revealed a differential flexibility and stability of the mutant compared to the wild-type PfFNR. Single-residue substitution within critical FNR motifs affects NADPH binding enzyme function; thus, G340S may impact enzyme catalysis, alter parasite fitness, and drug susceptibility. Experimental validations are required to confirm these predictions and assess PfFNR potential as both a biomarker and drug target in malaria control.恶性疟原虫（Plasmodium falciparum）作为致死性最强的疟原虫，仍在持续给撒哈拉以南非洲地区的医疗系统带来沉重负担。为减轻这一负担，青蒿素联合疗法（Artemisinin-based Combination Therapies，ACTs），尤其是蒿甲醚-苯芴醇（AL），仍是一线治疗方案。然而，青蒿素部分耐药性正在扩散，对必需的长效配伍药物苯芴醇（LM）施加了选择压力。尽管苯芴醇曾被认为不易产生耐药性，但一旦出现耐药性，其疗效仍可能受到损害。为阐明与苯芴醇相关的耐药标志物，我们的预实验已定位到伯氏疟原虫铁氧还蛋白NADP⁺还原酶（Ferredoxin NADP⁺ Reductase，FNR）的G332S突变，该突变对应恶性疟原虫中的G340S突变。PfFNR是一种关键的顶质体酶，在核心生物合成途径与药物活化过程中发挥核心作用。为探究G340S替换对蛋白结构与功能的影响，本研究采用了计算机生物信息学方法：通过AlphaFold3与I-TASSER构建高置信度的野生型与突变型PfFNR模型，使用AutoDock Vina与AlphaFold3进行分子对接，并借助GROMACS开展分子动力学（MD）模拟；通过ChimeraX完成结构可视化与相互作用图谱分析。结构分析结果显示，G340S突变会改变NADP⁺结合环的构象，破坏辅因子结合重叠位点处的氢键网络，并在不改变蛋白整体折叠构象的前提下降低辅因子结合亲和力。分子对接结果表明，NADP⁺与铁氧还蛋白的保守结合位点出现局部结构扰动，这可能影响电子传递过程。分子动力学模拟结果显示，与野生型PfFNR相比，突变型PfFNR的柔性与稳定性存在显著差异。关键FNR基序内的单氨基酸替换会影响NADPH结合与酶功能，因此G340S突变可能会改变酶的催化活性、寄生虫适合度与药物敏感性。后续需开展实验验证以确认上述预测，并评估PfFNR作为疟疾防控中生物标志物与药物靶点的潜力。