Thermodynamic Preference of Fe ²⁺ for a C-Terminal Coordination Site in Human Frataxin Revealed by Multiscale Simulations

Frataxin (FXN) is a mitochondrial metallochaperone whose deficiency causes Friedreich’s ataxia (FRDA), the most prevalent inherited ataxia, yet the thermodynamically preferred Fe2+ coordination site has not been determined at atomic resolution under dynamic conditions. Here, we present a multiscale computational investigation of Fe2+ coordination to human frataxin (PDB: 1EKG), integrating 3.4 μs of aggregate simulation time across unbiased molecular dynamics, well-tempered metadynamics, alchemical free energy perturbation (FEP), DFT cluster calculations (B3LYP-D3BJ/def2-TZVP), and force field sensitivity analysis. Our simulations identify a previously uncharacterized C-terminal coordination site (Pocket 2, lined by HIS183, GLU184, ASP199, and LYS208, with GLU184 and the LYS208 C-terminal carboxylate serving as first-shell ligands) computationally predicted to be thermodynamically preferred over the canonical acidic ridge by ΔΔG = −16.85 ± 9.5 kJ/mol; while the magnitude of this preference should be interpreted with caution given the known limitations of nonpolarizable FEP for divalent cations, the consistent direction across multiple independent methods supports Pocket 2 as a higher-affinity site. Both sites retain Fe2+ for 500 ns with a metadynamics-derived dissociation barrier of ∼115 kJ/mol, and the coordination geometry (first-shell RDF peak at 1.92 Å, exclusively O/N ligands; Mayer BVA = 0.099) is consistent with experimental XAS/EXAFS data. Fe2+ binding is associated with global conformational rigidification, narrowing of the conformational ensemble, and emergence of correlated motion between the C-terminal coordination region and the acidic ridge, supporting a working dual-site binding hypothesis that may provide a new structural framework for understanding frataxin’s metallochaperone function in Friedreich’s ataxia.