Biostructural Pathology of the Loss of the 225S-S282 Disulfide Bond in HFE: A Molecular Dynamic Study of the p.Arg226Gly HFE Variant

Mutations in the HFE gene may result in iron overload and can produce hereditary hemochromatosis. Most patients with hereditary hemochromatosis are homozygous for a single mutation of the HFE gene (p.Cys282Tyr). The molecular defect lies in the loss of the unique disulfide bridge present in the α3 domain of the protein, which is required for proper folding of HFE, trafficking to the cell surface and binding to β2-microglobulin. We have studied the impact of a new missense mutation (p.Arg226Gly) on the structure of the α3-domain of HFE by means of Molecular Dynamics (MD) calculations in explicit solvent.

Materials and Methods: A computer model structure of the α3 domain (i.e. 93 residues, Gln₂₀₅-Trp₂₉₇) was generated based on the X-Ray crystal structure. All MD simulations were performed with the SANDER module of the AMBER10 program suite using the Hornak et al. all-atom model force field (FF99SB parameters). Structural properties were analyzed with the PTRAJ module present within AmberTools1.2. The relative stability of the HFE^WT and HFE^R226G proteins was evaluated in terms of free energy of the relevant systems by post-processing the molecular dynamics trajectories with the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) technique.

The patient was a 45-year-old male from Brittany presenting with abdominal pain and asthenia. He had serum ferritin (SF) of 2325 microg.l⁻¹ (N: 10–300 microg.l⁻¹), and transferrin saturation (TSat) level of 49 % (N < 45 %). Molecular genetics analysis revealed a new C282Y/R226G compound heterozygote genotype. A 33 years old brother in the same compound heterozygous state had an elevated SF of 552 microg.l⁻¹ and a TSat level of 47%. No other known iron gene was mutated in those described as being responsible for hemochromatosis. The Arg226Gly substitution is located in the vicinity of the Cys225S-S282Cys disulfide bond. Our results show: i) that the p.Arg226Gly mutation S-S prevents the formation of the Cys225S-S282Cys disulfide bridge in increasing the distance between the 225–282 sulfur groups [d= 3.57 Å ± 0.21 for HFE^WT and 4.25 Å ± 0.52 for HFE^226G]; ii) consequently, many others local features of the protein, such as the solvation free energy and hydrophobicity are modified, although the global secondary structure of the α3 domain is conserved. This is confirmed altogether by the rmsd/rmsf plots and the superimposition of three-dimensional structures. This lack of global conformational modifications points out the structural adaptability and flexibility of proteins to adapt to missense mutations with minor local rearrangements. Analyses to control formation or deformation of salt-bridges were undergone as well. Surprisingly, Arg226 is not involved in any of new salt bridges on the contrary of its Arg224 neighbor. Instead of causing conformational disruption, losing the 225S-S282 disulfide bridge destabilizes the native state and modifies the stability of the protein. Instability is caused by the absence of internal water molecules in the vicinity of the mutation as evidenced on the rdfs curves. Compared to HFE^WT, the mutant presents two additional hydrophobic pockets, one positioned around residue 226, whereas the second one is located slightly above the two cysteine residues (225 and 282) conferring the mutant with additional properties.

The p.Arg226G substitution in HFE is one of the very rare natural examples of a loss of a disulfide bond without a cysteine substitution. As it is not possible to study extensively all naturally occurring mutations in proteins by using biochemical tests and physiological experiments including animal models, computer approaches represent a very useful alternative when the X ray structure is known for the wild type protein.

No relevant conflicts of interest to declare.