Electrostatic and Structural Bases of Fe2+ Translocation through Ferritin Channels

Abstract

Edited by F. Peter GuengerichFerritin molecular cages are marvelous 24-mer supramolecular architectures that enable massive iron storage (>2000 iron atoms) within their inner cavity. This cavity is connected to the outer environment by two channels at C3 and C4 symmetry axes of the assembly. Ferritins can also be exploited as carriers for in vivo imaging and therapeutic applications, owing to their capability to effectively protect synthetic non-endogenous agents within the cage cavity and deliver them to targeted tissue cells without stimulating adverse immune responses. Recently, X-ray crystal structures of Fe 2؉ -loaded ferritins provided important information on the pathways followed by iron ions toward the ferritin cavity and the catalytic centers within the protein. However, the specific mechanisms enabling Fe 2؉ uptake through wild-type and mutant ferritin channels is largely unknown. To shed light on this question, we report extensive molecular dynamics simulations, site-directed mutagenesis, and kinetic measurements that characterize the transport properties and translocation mechanism of Fe 2؉ through the two ferritin channels, using the wild-type bullfrog Rana catesbeiana H protein and some of its variants as case studies. We describe the structural features that determine Fe 2؉ translocation with atomistic detail, and we propose a putative mechanism for Fe 2؉ transport through the channel at the C3 symmetry axis, which is the only iron-permeable channel in vertebrate ferritins. Our findings have important implications for understanding how ion permeation occurs, and further how it may be controlled via purposely engineered channels for novel biomedical applications based on ferritin.Twenty-four-mer ferritins are ubiquitous iron storage proteins that share a common architecture: a protein nanocage (see Fig. 1A) assembled from subunits made up by a 4-helix bundle (helices H1-H4) structure completed by a short C-terminal helix, H5, and a long loop connecting helices H2 and H3. This protein shell surrounds an 8-nm inner cage connected to the external environment by two different types of channels: eight channels in correspondence with the four 3-fold (C3, see Fig. 1B) symmetry axes and six channels in correspondence with the three 4-fold (C4, see Fig. 1C) symmetry axes of the octahedral point symmetry of the cage (1, 2). Despite many similarities across ferritins expressed in different species, the interiors of the C3 and C4 channels are quite variable, showing substantial differences in terms of hydrophobicity/hydrophilicity and electric charge distributions when comparing vertebrate ferritins with those from plants, bacteria, or archaea (3, 4). In turn, the specific chemical nature of these channels directly affects their transport properties and determines the preferred pathways followed by ferrous ions from the exterior of the cage to the catalytic ferroxidase center within the internal cavity. In vertebrate ferritins, for example, the C4 channels (about 12 Å in length) are relatively narrow and mai...

Iron Biomineral Growth from the Initial Nucleation Seed in L‐Ferritin

“…The continuous negative patch corresponding to the clusters A–C might be designed to assist the initial steps of the biomineralization (Figures D and D); this patch is completely cancelled in the variant E60AE61AE64A, where the formation of the octa‐iron cluster does not occur. The present observations represent a further proof of the role played by electrostatic guidance in ferritin reactivity . On the basis of these results, the here observed octa‐iron moiety appears as a promising candidate of functional relevance.…”

“…Other Fe 3+ ions have been detected in HuLf and its triple variant E60AE61AE64A starting from 15 min exposure inside the threefold channels, which represent the iron entry channels in animal ferritins, and on the external protein surface in correspondence of a negative surface area, as detailed in the Supporting Information.…”

Routes of iron entry into, and exit from, the catalytic ferroxidase sites of the prokaryotic ferritin SynFtn

“…The route of iron transit has been elucidated for recombinantly expressed homopolymers of human H and frog M ferritin, largely due to the work of the groups of Theil, Turano and Mangani. [5][6][7][8][9][10] The peptide sequences of the ferritins of higher eukaryotes are highly conserved with respect to residues located close to the 3-fold channel ( Fig. S1 †), and both crystallographic data and disruption of this channel by site directed mutagenesis demonstrate that this is the route of iron entry into these proteins.…”

Structural Biology of Iron‐Binding Proteins by NMR Spectroscopy

“…By freezing ferritin crystals at different time intervals after exposure to a ferrous salt, it was possible to identify the iron binding ligands in the ferroxidase site (that were fully consistent with the NMR results) and to draw the path along which iron(II) ions move to reach the ferroxidase site , , , . The driving force for the observed intra‐cage iron trafficking has been analyzed by studies on a series of protein variants obtained by site directed mutagenesis that were investigated by integrating structural data, computational approaches, and kinetic measurements of the efficiency of ferroxidase and biomineralization activities , …”