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<p>FapR is a conserved transcriptional repressor that governs the fatty acid synthesis (FAS II) pathway in Gram-positive bacteria, including <i>Staphylococcus aureus</i>, by sensing intracellular malonyl-CoA (MCoA) levels through its effector-binding domain (EBD). Previous <i>in vivo</i> studies have demonstrated that simultaneous substitutions G111V and L132W within the EBD were lethal to <i>S. aureus</i>, even with exogenous fatty acid supplementation; however, the mechanistic basis of this phenotype has remained uncharacterised. Here, we delineate the structural and functional implications of these mutations using integrated <i>in silico</i> modelling and <i>in vitro</i> biochemical studies. AlphaFold 3-based structural prediction of the double mutant revealed overall conservation of the global fold (RMSD 0.532&#xa0;Å, relative to the wild-type crystal structure), but a pronounced constriction and distortion at the tunnel entrance through which the malonyl-phosphopantetheine arm of MCoA normally enters. Docking results confirmed that MCoA binds only superficially to the mutant, failing to penetrate the hydrophobic tunnel or form key hydrogen and electrostatic interactions. Recombinant double mutant FapR retained proper dimerisation and high-affinity DNA binding to the fapO operator. Intrinsic tryptophan fluorescence quenching assays demonstrated reduced affinity for MCoA relative to native FapR. Electrophoretic mobility shift assays further showed that, unlike the wild-type protein, MCoA does not disrupt DNA binding of the mutant repressor. Together, these findings indicate that obstruction of the tunnel entrance prevents productive effector engagement, thereby locking FapR in its DNA-bound conformation. This work elucidates why tunnel-entrance mutations are lethal and underscores FapR’s viability as a novel antistaphylococcal drug target.</p>

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Functional deregulation of FapR by tunnel-entry mutations reveal its vulnerability as an antistaphylococcal drug target

  • 期刊论文

  • 发表日期：

    2026年6月1日

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  • Amenti Ozukum,
  • Dipten Laskar,
  • Ekonthung Ezung,
  • Rajkrishna Mondal

摘要

FapR is a conserved transcriptional repressor that governs the fatty acid synthesis (FAS II) pathway in Gram-positive bacteria, including Staphylococcus aureus, by sensing intracellular malonyl-CoA (MCoA) levels through its effector-binding domain (EBD). Previous in vivo studies have demonstrated that simultaneous substitutions G111V and L132W within the EBD were lethal to S. aureus, even with exogenous fatty acid supplementation; however, the mechanistic basis of this phenotype has remained uncharacterised. Here, we delineate the structural and functional implications of these mutations using integrated in silico modelling and in vitro biochemical studies. AlphaFold 3-based structural prediction of the double mutant revealed overall conservation of the global fold (RMSD 0.532 Å, relative to the wild-type crystal structure), but a pronounced constriction and distortion at the tunnel entrance through which the malonyl-phosphopantetheine arm of MCoA normally enters. Docking results confirmed that MCoA binds only superficially to the mutant, failing to penetrate the hydrophobic tunnel or form key hydrogen and electrostatic interactions. Recombinant double mutant FapR retained proper dimerisation and high-affinity DNA binding to the fapO operator. Intrinsic tryptophan fluorescence quenching assays demonstrated reduced affinity for MCoA relative to native FapR. Electrophoretic mobility shift assays further showed that, unlike the wild-type protein, MCoA does not disrupt DNA binding of the mutant repressor. Together, these findings indicate that obstruction of the tunnel entrance prevents productive effector engagement, thereby locking FapR in its DNA-bound conformation. This work elucidates why tunnel-entrance mutations are lethal and underscores FapR’s viability as a novel antistaphylococcal drug target.