Opening protein pores with chaotropes enhances Fe reduction and chelation of Fe from the ferritin biomineral

Abstract

Iron is concentrated in ferritin, a spherical protein with a capacious cavity for ferric nanominerals of <4,500 Fe atoms. Global ferritin structure is very stable, resisting 6 M urea and heat (85°C) at neutral pH. Eight pores, each formed by six helices from 3 of the 24 polypeptide subunits, restrict mineral access to reductant, protons, or chelators. Protein-directed transport of Fe and aqueous Fe 3؉ chemistry (solubility Ϸ10 ؊18 M) drive mineralization. Ferritin pores are ''gated'' based on protein crystals and Fe chelation rates of wild-type (WT) and engineered proteins. Pore structure and gate residues, which are highly conserved, thus should be sensitive to environmental changes such as low concentrations of chaotropes. We now demonstrate that urea or guanidine (1-10 mM), far below concentrations for global unfolding, induced multiphasic rate increases in Fe 2؉ -bipyridyl formation similar to conservative substitutions of pore residues. Urea (1 M) or the nonconservative Leu͞Pro substitution that fully unfolded pores without urea both induced monophasic rate increases in Fe 2؉ chelation rates, indicating unrestricted access between mineral and reductant͞chelator. The observation of low-melting ferritin subdomains by CD spectroscopy (melting midpoint 53°C), accounting for 10% of ferritin ␣-helices, is unprecedented. The low-melting ferritin subdomains are pores, based on percentage helix and destabilization by either very dilute urea solutions (1 mM) or Leu͞Pro substitution, which both increased Fe 2؉ chelation. Biological molecules may have evolved to control gating of ferritin pores in response to cell iron need and, if mimicked by designer drugs, could impact chelation therapies in iron-overload diseases.

Gated pores in the ferritin protein nanocage

“…Electron transfer from reduced flavin to the ferric mineral is the rate limiting step in the analysis, which, based on comparisons of rates for different flavins, free and immobilized on agarose [19], appear to have limited access to the mineral. Studies with a variety of organic molecules into ferritin protein nanocage cavities shows a temperature dependence similar to that observed for pore unfolding by circular dichroism [3,21]. If contact between the mineral surface inside the ferritin nanocage and reduced flavin is increased by opening the gated pores in the protein, the rate of ferrous transfer out of the ferritin pores will increase (see Table 1 and Figs.…”

“…Since the protein pores in the ferritin nanocages control access between ferric mineral and reductants, the "patch clamp" assay for ferritins is the addition of the reductant to a solution of mineralized ferritin in the presence of a reporter for iron outside the nanocages. Formation rates of ferrous bipyridyl are the most common parameters measured for ferritin gated pore function, but since the reactions are usually carried out in air, a ferric chelator, such as desferal, can also be used as a reporter [3]. When the reaction conditions are constant, rate differences for the same protein under different conditions or for mutant proteins compared to wild type proteins, indicate changes in the protein nanocage itself (Table 1).…”

“…The presence of gates in the pores of ferritin nanocages has been recognized only recently from structure/function studies. Physiological ligands for ferritin gated pores are still to be identified, although the effects of physiological concentrations of urea appear to be an important clue [3].…”

“…The model is weakened by the absence of crystal structures of the mutant proteins, by the multiple sites for metal inhibitor binding in addition to the pores, and by studies suggesting other ferrous ion entry sites besides the gated pores [12,13]. In contrast, the model of ferrous ion transfer from the reduced mineral in the cavity through the gated pores is based on parallel structural studies in solution and crystals with functional studies in solution [3,14,15].…”

The role of lysosomes in iron metabolism and recycling

“…Apart from the autophagic pathway, some ferritin is also degraded by proteasomes and these two pathways may be able to partially compensate for each other (De Domenico et al, 2006, De Domenico et al, 2009, Zhang et al, 2010. A less well documented, but still possible mechanism of iron release from ferritin is by way of pores in the ferritin molecules (Liu et al, 2003, Takagi et al, 1998.…”