Peptides Selected for the Protein Nanocage Pores Change the Rate of Iron Recovery from the Ferritin Mineral

Abstract

Pores regulate access between ferric-oxy biomineral inside and reductants/chelators outside the ferritin protein nanocage to control iron demineralization rates. The pore helix/loop/ helix motifs that are contributed by three subunits unfold independently of the protein cage, as observed by crystallography, Fe removal rates, and CD spectroscopy. Pore unfolding is induced in wild type ferritin by increased temperature or urea (1-10 mM), a physiological urea range, 0.1 mM guanidine, or mutation of conserved pore amino acids. A peptide selected for ferritin pore binding from a combinatorial, heptapeptide library increased the rate of Fe demineralization 3-fold (p < 0.001), similarly to a mutation that unfolded the pores. Conjugating the peptide to Desferal (desferrioxamine B mesylate), a chelator in therapeutic use, increased the rates to 8-fold (p < 0.001). A second pore binding peptide had the opposite effect and decreased the rate of Fe demineralization 60% (p < 0.001). The peptides could have pharmacological uses and may model regulators of ferritin demineralization rates in vivo or peptide regulators of gated pores in membranes. The results emphasize that small peptides can exploit the structural plasticity of protein pores to modulate function.Pores in ferritin protein cages are an example of pores in molecular or ionic barriers; other examples are pores in membranes. In ferritin, pores control reactions between reductants outside the protein and the ferric mineral inside. As with many gated pores, ferritin pores are formed by ␣-helices in multiple subunits that surround the ion path and are modulated by unfolding (1, 2) (see Fig. 1). Ferritin pores are arranged symmetrically around the protein cage, eight for 24 subunit maxi-ferritins and four for 12 subunit maxi-ferritins, also called Dps proteins (2, 3).Ferritins concentrate Fe for biological use and also detoxify Fe/O 2 or H 2 O 2 in the ferric/oxy biomineral inside the proteins of Archaea, bacteria, and Eukaryota, including higher plants and animals. Fe ions destined to enter the mineral appear to reach the ferroxidase coupling site through the pores for the first step in biomineralization (2, 4 -7). The critical roles of ferritins are illustrated by lethality of deletions in mice, neurological effects of mutations in humans, and pathogen responses to host-released oxidants (3,8,9). In addition, dual genetic regulatory systems with DNA (antioxidant-response elements) enhancers linking ferritin regulation to antioxidant response proteins (10, 11) and mRNA "promoters" (iron-response element) linking ferritin regulation to Fe trafficking proteins emphasize the central role of ferritin in Fe and oxygen metabolism. Finally, the ferritin Fe reactions with O 2 or H 2 O 2 (2, 4 -7) and the presence of ferritin in anaerobic archaea (12) suggest an ancient role for ferritins in the transition to aerobic metabolism.The rates of Fe transport from the ferritin mineral through the nanocage channels and pores to chelators on the outside of the nanocage are initiated by...

Biology of ferritin in mammals: an update on iron storage, oxidative damage and neurodegeneration

“…Increasing temperature, physiological concentration of chaotropic reagents or site-directed mutagenesis of specific residues of tadpole M-ferritin (Leu-110, Arg-72 an d Asp-122) have been shown to dramatically increase the in vitro rate of ferritin iron release by altering the stability of the 3-fold channels. Furthermore, the screening of a combinatorial library brought to the identification of peptides capable of binding to ferritin, opening or closing the pores and thus modulating the rate of iron release (Liu et al 2007). This finding led to the speculative proposal of regulatory molecules controlling the breathing of the gated pores in vivo.…”

Brain Iron Homeostasis: From Molecular Mechanisms To Clinical Significance and Therapeutic Opportunities

“…Available information suggests that a cytosolic iron chaperone Poly (rC)-binding protein 1 binds cytosolic iron for delivery to ferritin (398). Poorly characterized ferritin-binding proteins, amino acids, and small molecules regulate the release of iron from ferritin, supporting the gated pore model (273,274,427). Stored iron is released in response to low intracellular iron, thus maintaining equilibrium between ferritin iron and the labile iron pool.…”

A New Role for Heme, Facilitating Release of Iron from the Bacterioferritin Iron Biomineral

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