Fe(2+) substrate transport through ferritin protein cage ion channels influences enzyme activity and biomineralization

Abstract

Ferritins, complex protein nanocages, form internal iron-oxy minerals (Fe2O3·H2O), by moving cytoplasmic Fe(2+) through intracage ion channels to cage-embedded enzyme (2Fe(2+)/O2 oxidoreductase) sites where ferritin biomineralization is initiated. The products of ferritin enzyme activity are diferric oxy complexes that are mineral precursors. Conserved, carboxylate amino acid side chains of D127 from each of three cage subunits project into ferritin ion channels near the interior ion channel exits and, thus, could direct Fe(2+) movement to the internal enzyme sites. Ferritin D127E was designed and analyzed to probe properties of ion channel size and carboxylate crowding near the internal ion channel opening. Glu side chains are chemically equivalent to, but longer by one -CH2 than Asp, side chains. Ferritin D127E assembled into normal protein cages, but diferric peroxo formation (enzyme activity) was not observed, when measured at 650 nm (DFP λ max). The caged biomineral formation, measured at 350 nm in the middle of the broad, nonspecific Fe(3+)-O absorption band, was slower. Structural differences (protein X-ray crystallography), between ion channels in wild type and ferritin D127E, which correlate with the inhibition of ferritin D127E enzyme activity include: (1) narrower interior ion channel openings/pores; (2) increased numbers of ion channel protein-metal binding sites, and (3) a change in ion channel electrostatics due to carboxylate crowding. The contributions of ion channel size and structure to ferritin activity reflect metal ion transport in ion channels are precisely regulated both in ferritin protein nanocages and membranes of living cells.

Figure 1. (A-D): Fe²⁺ traffic into the buried enzyme sites of ferritin protein cages (eukaryotic model: frog M)

Ferritin enzyme activity (Fe²⁺/O₂ oxidoreductase) requires Fe²⁺ entry into the ferritin protein nanocage from the external environment, which are connected by 8 ion channels around the 3-fold symmetry axes of 24 subunit ferritin cages A. A ferritin nanocage: self-assembled from 24 subunits, there are 8 ion channels around the 3-fold symmetry axes (red helices) for delivery of iron to 24 di-iron active sites (one/subunit); one arrow point to 1 of 24 enzyme sites with 2 substrate ions (orange spheres) and another arrow points to an ion channel where Fe²⁺ substrate enters or Fe²⁺ from dissolved, caged ferritin mineral exits. B. Symmetric distribution of incoming metals ions from D127, at the inner ion channel exits (inside view); arrows indicate connections between an interior ion channel and ferritin enzymatic (2Fe²⁺/O₂ oxidoreductase) sites; oxidoreductase site residues- red-orange. C. A line of Mg²⁺ ions (green spheres), bind to conserved residues in ferritin ion channels, around each 3-fold symmetry axis of the protein cage. D. Side chains from three conserved E130 residues (one from each of three protein cage subunits forming the ion channels at the protein cage 3-fold symmetry axes) create a constriction mid-way in ferritin ion channels. The diameter at a ferritin protein channel constriction is smaller than the 6.9 Å diameter of [Fe (H₂O)₆]²⁺ ; fully or partially dehydrated Fe²⁺ ions, which have diameters smaller than the 6.9 Å diameter at the ferritin ion channel constrictions, suggest partial or full dehydration of transiting Fe²⁺ ions.

Figure 2. (A-D): Ferritin enzyme activity (Fe²⁺/O₂ oxidoreduction) is inhibited in ferritin D127E, an amino acid substitution near the inner exits of the ion channels: single and multiple turnover experiments

A. Progress curves at A_650nm for formation/decay of DFP intermediate. B. Progress curves of A_350nm for formation [Fe³⁺-O]_x species. WT and variant ferritin (4.16 μM nanocages) in 200 mM MOPS, 200 mM NaCl at pH 7.0 and 200 μM FeSO₄ (48 Fe/cage) in 1.0 mM HCl were mixed in equal volumes at 20°C in a stopped-flow spectrophotometer. (See Experimental Procedures.) C. and D. Progress curves for WT and D127E ferritins with 480 Fe/cage at A_650nm and A_350nm; conditions are same as in A and B except the final protein concentration was decreased to half (from 2.08 to 1.04 μ) in order to avoid oxygen as limiting factor. NOTE: the most rapid period of change occurs in less than one second after mixing.

Figure 3. Mineral dissolution increased in ferritin D127E, substitutions in the three subunits forming ferritin ion channels

Mineral dissolution requires access between the protein caged- ferric iron mineral and reductants, NADH/FMN and, as measured here, between Fe²⁺ dissolved from the ferritin ferric mineral, and the chelator. A. The iron release kinetics were monitored at 25°C by measuring the amount of Fe²⁺- (2,2′-bipyridyl)₃ outside the ferritin nanocage (absorbance change at 522 nm) (See Experimental Procedures). B. Effect of D127E substitution in ferritin protein cages on the percentage of iron released after 30 min. The final concentrations of the solutions were 250 μM caged ferric mineral, 2.5 mM NADH, 2.5 mM FMN, and 2.5 mM bipyridyl, in 0.1 M MOPS and 0.1 M NaCl, pH 7.0. * for p-value <0.0001 computed against WT. The data shown are averages of results from 4-6 independent experiments, using two different preparations of each protein. Errors are the standard deviation.

Figure 4. (A-F): Differences and similarities in ion distribution in ferritin ion channels helices in WT and D127E ferritin protein cages

Ion channels form around the 3-fold axes of ferritin protein cages. The channels consist of segments from two α helices (α3 and α4) in the four α-helix bundles that form each subunit of ferritin protein cages. Helices are shown as ribbon representation and waters as small red spheres. A-C. Side views of Mg²⁺ bound ferritin cocrystal structures. A. WT ferritin structure with Mg²⁺ (PDB code: 3KA3): carbon and oxygen atoms in white and red stick, respectively; Mg²⁺ as pale green spheres; Cl^- ions as yellow spheres. B. D127E ferritin structure (PDB code: 4LPM) carbon, and oxygen atoms in white and red stick, respectively; Mg²⁺ ions as dark green spheres; Cl^- as yellow spheres. C. Superposition of WT and D127E Mg²⁺ bound ferritin cocrystal structures. D-F. Side views of Co²⁺ and Mg²⁺ bound ferritin cocrystal structures. D. WT ferritin structure (PDB code: 3KA4): carbon and oxygen atoms in white and red stick, respectively; Co²⁺ as pale pink spheres; Cl^- as yellow spheres. E. D127E ferritin structure (PDB code: 4LPN): carbon and nitrogen atoms in blue and stick, respectively, Co²⁺ as dark pink spheres, Cl^- as yellow spheres. F. Superimposition of WT and D127E Co²⁺ and Mg²⁺ bound ferritin cocrystal structures.

Figure 5. (A-F): Structural and conformational comparisons of Fe²⁺ transfer and Fe²⁺/O₂ oxidoreductase sites in WT and the D127E ferritin protein cages

E57 and E136 are transfer residues. Helices are shown as ribbon representation and waters are shown as small red spheres. A-C. Side views of the Mg²⁺ bound ferritin cocrystal structures A. WT ferritin structure (PBD code: 3KA3), where carbon, nitrogen and oxygen atoms in white, blue and red stick, respectively, Mg²⁺ ions represented as pale green spheres, and alternate WT side-chain conformations carbons are colored orange. B. D127E ferritin structure, where carbon atoms are shown in carbon, nitrogen and oxygen atoms in dark blue, blue and red stick, respectively, Mg²⁺ ions represented as dark green spheres, and the extra water characteristic of the D127E ferritin variant is a yellow sphere. C. Superposition of WT and D127E Mg²⁺ bound structures. D-F. Side view of Co²⁺ bound ferritin cocrystal structures. D. WT ferritin structure (PDB code: 3KA4), carbon, nitrogen and oxygen atoms in white, blue and red stick, respectively, Co²⁺ ions represented as pale pink spheres, and alternate side-chain conformations carbons are colored orange. E. D127E ferritin structure, where carbon, nitrogen and oxygen atoms in dark blue, blue and red stick, respectively, Co²⁺ ions represented as dark pink spheres, and alternate D127E side-chain conformations carbons are colored yellow. F. Superimposition of WT and D127E ferritin Co²⁺ bound structures.