Moving Fe
            <sup>2+</sup>
            from ferritin ion channels to catalytic OH centers depends on conserved protein cage carboxylates

Behera, Rabindra K.; Theil, Elizabeth C.

doi:10.1073/pnas.1318417111

Cited by 76 publications

(142 citation statements)

References 29 publications

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“…These results, although they have to be considered with caution due to inherent approximations in our modeling, do support the view that ferrous iron does travel as aqua ions from the outer environment to the ferroxidase catalytic centers within the ferritin cage, as observed in X-ray crystal structures. Additionally, the present kinetics data exclude any involvement of Cys-126, a solvent-exposed residue located on the ferritin shell close to the C3 channel entrance, in the iron uptake process, contrary to previous suggestions (23). Concerning the 4-fold channel, the larger number of negative charges and the wider pore size of C4TM with respect to C3WT account for the higher iron uptake rate observed in the former ferritin variant (8).…”

Section: Discussioncontrasting

confidence: 94%

See 1 more Smart Citation

Electrostatic and Structural Bases of Fe2+ Translocation through Ferritin Channels

Chandramouli

Bernacchioni

Maio

et al. 2016

Journal of Biological Chemistry

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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...

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Section: Discussioncontrasting

confidence: 94%

“…This observation appears in very good agreement with free energy calculations reported above. In addition, other studies have reported the possible interaction of metal ions with Cys-126 residues located at the entrance of C3 channels (22), and the interaction with Fe 2ϩ has also been inferred (23). Our data (supplemental Figs.…”

Section: Resultssupporting

confidence: 82%

Electrostatic and Structural Bases of Fe2+ Translocation through Ferritin Channels

Chandramouli

Bernacchioni

Maio

et al. 2016

Journal of Biological Chemistry

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“…According to the X-ray data (PDB: 4LPJ, 4LQJ, 4LYX, 4LYU, 4LQV, 4LQN, 3RGD) [8,12], the permeability to iron of the ferroxidase site arises from a set of amino acids (E136, E57, H54) located in the close proximity of the catalytic center and facing the inner cavity of the cage. Substitution of conserved cage carboxylates E136 and/or E57 with Ala, slows down the catalytic reaction in bullfrog M ferritin, as recently demonstrated [11], supporting the idea that the translocation of cations from the inner cavity to the bundle interior is again driven by electrostatic attractions exerted by clusters of negatively charged residues.…”

Section: Discussionsupporting

confidence: 70%

“…The analysis of the available X-ray structures of animal ferritins with non-native divalent cations (Cu 2+ , Co 2+ , Zn 2+ , Mg 2+ ) has revealed the presence of several amino acid side chains that can potentially act as metal ion ligands [8][9][10]; their number is larger than what needed to bind the preoxidation diferrous species and the diferric-peroxo (DFP, hereafter) and ferric-oxo/hydroxo products. The several potential metal binding amino acids in the same area have been proposed to play a role in regulating the access of the iron substrate to the reaction center [8][9][10][11].…”

Section: Introductionmentioning

confidence: 99%

Is His54 a gating residue for the ferritin ferroxidase site?

Bernacchioni¹,

Ciambellotti²,

Theil³

et al. 2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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“…5) and is followed by oxidation at specific, yet unobserved, nucleation sites that facilitate the subsequent growth of the mineral (2,4,21,22). Using X-ray crystallography, we detected iron bound at inner cage sites of L-subunits of recombinant homopolymeric human ferritin in the form of (μ 3 -oxo)Tris[(μ 2 -peroxo)(μ 2 -glutamato-κO:κO′)](glutamato-κO)(diaquo)triiron(III) anion; a very similar arrangement was here observed in HoLf.…”

Section: Discussionmentioning

confidence: 99%

Chemistry at the protein–mineral interface in L-ferritin assists the assembly of a functional (μ ³ -oxo)Tris[(μ ² -peroxo)] triiron(III) cluster

Pozzi

Ciambellotti

Bernacchioni

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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X-ray structures of homopolymeric L-ferritin obtained by freezing protein crystals at increasing exposure times to a ferrous solution showed the progressive formation of a triiron cluster on the inner cage surface of each subunit. After 60 min exposure, a fully assembled (μ 3 -oxo)Tris[(μ 2 -peroxo)(μ 2 -glutamato-κO:κO′)](glutamato-κO)(diaquo)triiron(III) anionic cluster appears in human L-ferritin. Glu60, Glu61, and Glu64 provide the anchoring of the cluster to the protein cage. Glu57 shuttles incoming iron ions toward the cluster. We observed a similar metallocluster in horse spleen L-ferritin, indicating that it represents a common feature of mammalian L-ferritins. The structures suggest a mechanism for iron mineral formation at the protein interface. The functional significance of the observed patch of carboxylate side chains and resulting metallocluster for biomineralization emerges from the lower iron oxidation rate measured in the E60AE61AE64A variant of human L-ferritin, leading to the proposal that the observed metallocluster corresponds to the suggested, but yet unobserved, nucleation site of L-ferritin.L-ferritin | metallocluster | nucleation site | biomineralization | X-ray

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Moving Fe ²⁺ from ferritin ion channels to catalytic OH centers depends on conserved protein cage carboxylates

Cited by 76 publications

References 29 publications

Electrostatic and Structural Bases of Fe2+ Translocation through Ferritin Channels

Electrostatic and Structural Bases of Fe2+ Translocation through Ferritin Channels

Is His54 a gating residue for the ferritin ferroxidase site?

Chemistry at the protein–mineral interface in L-ferritin assists the assembly of a functional (μ ³ -oxo)Tris[(μ ² -peroxo)] triiron(III) cluster

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Moving Fe 2+ from ferritin ion channels to catalytic OH centers depends on conserved protein cage carboxylates

Cited by 76 publications

References 29 publications

Electrostatic and Structural Bases of Fe2+ Translocation through Ferritin Channels

Electrostatic and Structural Bases of Fe2+ Translocation through Ferritin Channels

Is His54 a gating residue for the ferritin ferroxidase site?

Chemistry at the protein–mineral interface in L-ferritin assists the assembly of a functional (μ 3 -oxo)Tris[(μ 2 -peroxo)] triiron(III) cluster

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Product

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Moving Fe ²⁺ from ferritin ion channels to catalytic OH centers depends on conserved protein cage carboxylates

Chemistry at the protein–mineral interface in L-ferritin assists the assembly of a functional (μ ³ -oxo)Tris[(μ ² -peroxo)] triiron(III) cluster