Gradual Modification of Ferritin 4-Fold Pore Promotes Cage Instability, Fe ²⁺ Exit, and Iron-Induced Protein Precipitation

Ferritins are symmetrical, hollow nanocaged iron storage proteins, which sequester and concentrate iron as a hydrated ferric oxyhydroxide (ferrihydrite) biomineral. Spontaneous self-assembly of 24 subunits in eukaryotic ferritins leads to the formation of symmetrical pores, i.e., eight hydrophilic 3-fold pores and six hydrophobic 4-fold pores. However, unlike the relatively wider 3-fold pores, which drive Fe²⁺ inside, the functions of narrow 4-fold pores are relatively understudied. The current work investigates the role of 4-fold pores by gradual alterations of specific amino acids using site-directed mutagenesis, structural analysis by X-ray crystallography, and solution-based kinetic studies. Increasing the negative charge in ferritin 4-fold pore retained the cage integrity and iron-loading capability despite altering the pore structure/size/electrostatics. As additional substitutions accumulated within the same channel, the aperture widens further and the electrostatic environment became progressively more acidic around the pore lining. These gradual alterations resulted in an enhanced rate of iron mobilization (up to ∼10-fold), possibly due to remodeling of the 4-fold (C4) pore, leading to a progressive increase in 4-fold pore diameter. Moreover, these modifications decreased cage stability, both thermally (∼5 °C) and chemically (∼3-4 folds), and increased iron-induced ferritin precipitation, similar to the case of neuroferritinopathy.