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Polymers (Basel)
2022 Dec 08;1424:. doi: 10.3390/polym14245378.
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Structural and Functional Insights into the Roles of Potential Metal-Binding Sites in Apostichopus japonicus Ferritin.
Wu Y
,
Huo C
,
Ming T
,
Liu Y
,
Su C
,
Qiu X
,
Lu C
,
Zhou J
,
Li Y
,
Zhang Z
,
Han J
,
Feng Y
,
Su X
.
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Ferritin is widely acknowledged as a conservative iron storage protein found in almost all living kingdoms. Apostichopus japonicus (Selenka) is among the oldest echinoderm fauna and has unique regenerative potential, but the catalytic mechanism of iron oxidation in A. japonicus ferritin (AjFER) remains elusive. We previously identified several potential metal-binding sites at the ferroxidase center, the three- and four-fold channels in AjFER. Herein, we prepared AjFER, AjFER-E25A/E60A/E105A, AjFER-D129A/E132A, and AjFER-E168A mutants, investigated their structures, and functionally characterized these ferritins with respect to Fe2+ uptake using X-ray techniques together with biochemical analytical methods. A crystallographic model of the AjFER-D129A/E132A mutant, which was solved to a resolution of 1.98 Å, suggested that the substitutions had a significant influence on the quaternary structure of the three-fold channel compared to that of AjFER. The structures of these ferritins in solution were determined based on the molecular envelopes of AjFER and its variants by small-angle X-ray scattering, and the structures were almost consistent with the characteristics of well-folded and globular-shaped proteins. Comparative biochemical analyses indicated that site-directed mutagenesis of metal-binding sites in AjFER presented relatively low rates of iron oxidation and thermostability, as well as weak iron-binding affinity, suggesting that these potential metal-binding sites play critical roles in the catalytic activity of ferritin. These findings provide profound insight into the structure-function relationships related to marine invertebrate ferritins.
Figure 1. Characterization of the AjFER, MF, M3, and M4 proteins: (A) CD spectra of the AjFER, MF, M3, and M4 proteins; (B) DLS analyses of the AjFER, MF, M3, and M4 proteins; (C–F) TEM images of the AjFER, MF, M3, and M4 proteins.
Figure 2. The crystal structure of the M3 protein: (A) overall structure view from the outside of the three-fold channel; (B) a single subunit of the M3 protein viewed from a ring composed of the D129A/E132A substitutions in the three-fold channel; (C) stereo view of the M3 protein nanocage; (D) schematic overview of incoming metal ions moving via the three-fold channel from outside the M3 protein cage. The arrows indicate the connections from the three-fold channel toward ferroxidase sites in which yellow and light-blue spheres represent iron and cadmium ions, respectively.
Figure 3. Electrostatic surface potential and nanoscale analyses along the three-fold channel of the M3 protein. (A) Overall view of the electrostatic surface potential from outside to inside the cage in the three-fold channel. Comparison of the electrostatic surface potential distribution views at the whole three-fold channel in (B) AjFER and (C) M3 proteins. The potential scale is rendered from −50 to +50 kTe−1 from red to blue. A comparative analysis of the diameter at the three-fold channel in the (D) AjFER and (E) M3 proteins.
Figure 4. Stereo view and structural superposition of the metal ion coordination environment at the ferroxidase center and the three-fold channel in the AjFER and M3 proteins. Metal ion coordination environment at the three-fold channel of the (A) AjFER (PDB code: 7VHR) and (B) M3 (PDB code: 7Y74) proteins. (C) Superposition of the metal ion coordination environment of the three-fold channel between the AjFER and M3 proteins. The green and light blue spheres represent the magnesium and cadmium ions, respectively, while the red spheres represent water molecules. The metal ion coordination environment at the ferroxidase center in the (D) AjFER and (E) M3 proteins. (F) Superposition of the metal ion coordination environment of the ferroxidase center between the AjFER and M3 proteins. Mg2+, Cd2+ and Fe2+ ions are indicated by green, light blue, and orange spheres, respectively, while the water molecules are represented by red spheres.
Figure 5. SAXS analysis of AjFER and its variants: (A) experimental SAXS scattering curves of the protein samples; (B) Kratky plot analysis; (C) pair distance distribution curves of the protein samples. Overlay of the filtered and averaged (gray) envelopes (calculated from the SAXS data) of the (D) AjFER, (E) MF, (F) M3, and (G) M4 proteins with the crystal structure of AjFER (PDB code: 7VHR).
Figure 6. Iron uptake analysis of AjFER and its variants. (A) Iron oxidation curves of the protein samples. The oxidation of Fe2+ to Fe3+ ions was monitored by an increase in the absorbance at 315 nm. (B) Determination of iron content among protein samples: Fe2+-loaded AjFER (AjFER+Fe2+), Fe2+-loaded MF protein (MF+Fe2+), Fe2+-loaded M3 protein (M3+Fe2+), and Fe2+-loaded M4 protein (M4+Fe2+). The molar ratios of iron ions vs. ferritin (Fe/Ftn) were calculated by ICP–MS and the ferritin content per unit volume was determined using a BCA kit. Significant differences are indicated by * (p < 0.05), or *** (p < 0.001).
Figure 7. Interactions of AjFER and its variants with Fe2+ ions analyzed by (A) CD and (B) MST. For MST analysis, the error bars represent the mean ± SD in accordance with three independent measurements. Both binding curves and Kd values are shown.
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