Crystal structure of plant ferritin reveals a novel metal binding site that functions as a transit site for metal transfer in ferritin

Abstract

Ferritins are important iron storage and detoxification proteins that are widely distributed in living kingdoms. Because plant ferritin possesses both a ferroxidase site and a ferrihydrite nucleation site, it is a suitable model for studying the mechanism of iron storage in ferritin. This article presents for the first time the crystal structure of a plant ferritin from soybean at 1.8-A resolution. The soybean ferritin 4 (SFER4) had a high structural similarity to vertebrate ferritin, except for the N-terminal extension region, the C-terminal short helix E, and the end of the BC-loop. Similar to the crystal structures of other ferritins, metal binding sites were observed in the iron entry channel, ferroxidase center, and nucleation site of SFER4. In addition to these conventional sites, a novel metal binding site was discovered intermediate between the iron entry channel and the ferroxidase site. This site was coordinated by the acidic side chain of Glu(173) and carbonyl oxygen of Thr(168), which correspond, respectively, to Glu(140) and Thr(135) of human H chain ferritin according to their sequences. A comparison of the ferroxidase activities of the native and the E173A mutant of SFER4 clearly showed a delay in the iron oxidation rate of the mutant. This indicated that the glutamate residue functions as a transit site of iron from the 3-fold entry channel to the ferroxidase site, which may be universal among ferritins.

FIGURE 1.

Amino acid sequences of the plant ferritin, SFER4, and other ferritin from vertebrate and bacteria. The sequences of SFER4 (GenBank number AB062756), human H (HuHF, M11146), human L (HuLF, M11147), bullfrog H (BfHF, M15655), and two E. coli ferritins (EcBFR (Swiss Prot P0ABD3) and EcFTN (GenBank number X53513)) are shown. Conserved ferroxidase center, putative nucleation center, and transit site discussed in this study are shown boxed. The letters F, N, and T on the boxed sequences indicate the ferroxidase site, nucleation site, and transit site, respectively.

FIGURE 2.

Ribbon diagram of the plant ferritin, SFER4. a and b, the whole oligomer viewed down a 3-fold and a 4-fold symmetry axis, respectively. The N-terminal extension peptide forms a loop and a short helix lies on the neighboring 3-fold symmetry mate subunit. c, stereoview of a superimposition of the soybean ferritin SFER4 subunit (blue) on the human ferritin H chain (red, PDB accession code 2FHA).

FIGURE 3.

a, positions of the three calcium binding sites, 3-fold symmetry axis, ferroxidase site, and novel intermediate site coordinated by Glu¹⁷³ in the ferritin shell. The calcium atoms are shown as red balls. Residues forming a metal binding site of the 3-fold axis, a novel “transit site,” and ferroxidase center are shown in yellow, cyan, and green, respectively. b, the structure of the deduced metal ion entry channel penetrating along the 3-fold symmetry axis and metal binding site in the channel. This channel is lined with hydrophilic side chains of Asp¹⁶⁴, Glu¹⁶⁷, and Thr¹⁶⁸, and two calcium ions per channel are seen (in red). Waters ligated to the calcium ions or hydrogen bonded to the side chains are shown in blue. Blue broken lines, hydrogen bonds; black broken lines, metal coordination bonds. The channels formed by chain L (shown in “yellow”), M (purple), and T (beige) are represented from the view perpendicular to the axis (stereo view).

FIGURE 4.

Inter-subunit interactions around the 4-fold symmetry axis of the SFER4. The channels are represented with two different orientations: (a) aligned on the 4-fold axis and (b) perpendicular to the axis (stereo view). The electron densities of the four lined histidines contoured at 1.5 σ are shown as a mesh in a. Blue broken lines, electrostatic interaction of histidine side chains.

FIGURE 5.

Metal binding site in the ferroxidase center of the SFER4 (a) and its variant E173A (b). A metal binding site positioned near the center (described below) is also shown a. The atom color code is black for calcium and gray for oxygen of water. Gray broken lines, hydrogen bonds; black broken lines, metal coordination bonds.

FIGURE 6.

A novel metal binding site coordinated by Glu¹⁷³. A novel metal binding site is represented by coordination residues, Glu¹⁷³ and Thr¹⁶⁸, with the electron density (2|F_o| − |F_c|) contoured at 1.5σ shown as a mesh. The atom color code is black for calcium and gray for oxygen of water. Black broken lines indicate the metal coordination bonds.

FIGURE 7.

Effect of E173A mutation on iron oxidation rates of plant ferritin SFER4. Progress curves of μ-oxo-bridged Fe(III) generation monitored at 310 nm upon the addition of ferrous iron sulfate ammonium to the SFER4 and its E173A variant at a protein concentration of 1 μm and iron concentrations of 20 (a) and 100 (b) μm. The formation of μ-oxo-bridged Fe(III) was calculated from absorbance at 310 nm. Data are given as the mean ± S.D. of at least three individual experiments.

FIGURE 8.

The hydrophilic route from the 3-fold channel to the ferroxidase site of the E173A variant (stereo view). This figure is the view from the inner cavity side. The ferroxidase site (Glu⁵⁶, Glu⁹¹, His⁹⁴, Glu¹⁴⁰, and Gln¹⁷⁴) of chain G and the 3-fold channel composed of chains G, F, and O of the E173A variant are illustrated. The 3-fold channel is lined with Asp¹⁶⁴ and Glu¹⁶⁷ of the three chains (left side). The hydrophilic route emerged because of the substitution (E173A) and the kink of the D-helix in Tyr¹⁷⁰. The calcium atoms and water molecules are shown as black and gray balls, respectively.