Structural and Biochemical Characterization of Silver/Copper Binding by Dendrorhynchus zhejiangensis Ferritin.

Ferritin with a highly symmetrical cage-like structure is not only key in the reversible storage of iron in efficient ferroxidase activity; it also provides unique coordination environments for the conjugation of heavy metal ions other than those associated with iron. However, research regarding the effect of these bound heavy metal ions on ferritin is scarce. In the present study, we prepared a marine invertebrate ferritin from Dendrorhynchus zhejiangensis (DzFer) and found that it could withstand extreme pH fluctuation. We then demonstrated its capacity to interact with Ag+ or Cu2+ ions using various biochemical and spectroscopic methods and X-ray crystallography. Structural and biochemical analyses revealed that both Ag+ and Cu2+ were able to bind to the DzFer cage via metal-coordination bonds and that their binding sites were mainly located inside the three-fold channel of DzFer. Furthermore, Ag+ was shown to have a higher selectivity for sulfur-containing amino acid residues and appeared to bind preferentially at the ferroxidase site of DzFer as compared with Cu2+. Thus, it is far more likely to inhibit the ferroxidase activity of DzFer. The results provide new insights into the effect of heavy metal ions on the iron-binding capacity of a marine invertebrate ferritin.

Structural Insights Into the Effects of Interactions With Iron and Copper Ions on Ferritin From the Blood Clam Tegillarca granosa.

In addition to its role as an iron storage protein, ferritin can function as a major detoxification component in the innate immune defense, and Cu2+ ions can also play crucial antibacterial roles in the blood clam, Tegillarca granosa. However, the mechanism of interaction between iron and copper in recombinant Tegillarca granosa ferritin (TgFer) remains to be investigated. In this study, we investigated the crystal structure of TgFer and examined the effects of Fe2+ and Cu2+ ions on the TgFer structure and catalytic activity. The crystal structure revealed that TgFer presented a typically 4-3-2 symmetry in a cage-like, spherical shell composed of 24 identical subunits, featuring highly conserved organization in both the ferroxidase center and the 3-fold channel. Structural and biochemical analyses indicated that the 4-fold channel of TgFer could be serviced as potential binding sites of metal ions. Cu2+ ions appear to bind preferentially with the 3-fold channel as well as ferroxidase site over Fe2+ ions, possibly inhibiting the ferroxidase activity of TgFer. Our results present a structural and functional characterization of TgFer, providing mechanistic insight into the interactions between TgFer and both Fe2+ and Cu2+ ions.

Integrative proteomics and metabolomics profiling of the protective effects of Phascolosoma esculent ferritin on BMSCs in Cd(II) injury.

Ferritin is the major intracellular iron storage protein and is essential for iron homeostasis and detoxification. Cadmium affects cellular homeostasis and induces cell toxicity via sophisticated mechanisms. Here, we aimed to explore the mechanisms of cytoprotective effect of Phascolosoma esculenta ferritin (PeFer) on Cd(II)-induced bone marrow mesenchymal stem cell (BMSC) injury. Herein, the effects of different treated groups on apoptosis and cell cycle were assessed using flow cytometric analysis. We further investigated the alterations of the three groups using integrative 2-DE-based proteomics and 1H NMR-based metabolomics profiles. The results indicate that PeFer reduces BMSC apoptosis induced by Cd(II) and delays G0/G1 cell cycle progression. A total of 19 proteins and 70 metabolites were significantly different among BMSC samples of the three groups. Notably, multiomics analysis revealed that Cd(II) might perturb the ER stress-mediated apoptosis pathway and disrupt biological processes related to the TCA cycle, amino acid metabolism, purine and pyrimidine metabolism, thereby suppressing the cell growth rate and initiating apoptosis; however, the addition of PeFer might protect BMSCs against cell apoptosis to improve cell survival by enhancing energy metabolism. This study provides a better understanding of the underlying molecular mechanisms of the protective effect of PeFer in BMSCs against Cd(II) injury.

Structural comparison of two ferritins from the marine invertebrate Phascolosoma esculenta.

For marine invertebrates with no adaptive immune system, ferritin is a major intracellular iron-storage protein with a critical role in innate immunity. Here, we present the crystal structures of two novel ferritins [Fer147 and Phascolosoma esculenta ferritin (PeFer)] from the marine invertebrate P. esculenta, which resides in muddy-bottom coastal regions. Fer147 and PeFer exhibit the 4-3-2 symmetry of cage-like hollow shells containing 24 subunits, similar to other known ferritins. Fer147 and PeFer contain both the conserved ferroxidase center and threefold channels. Subtle structural differences in the putative nucleation sites suggest possible routes of metal ion movement in the protein shells. However, the marked variation in the electrostatic potential of the threefold channels in Fer147 and the fourfold channels in PeFer suggests significant diversity between Fer147 and PeFer in terms of metal ion aggregation and cation exclusion. In summary, the presented crystal structures may serve as references for studies of the iron-storage mechanism of additional ferritins from marine invertebrates.

Structure determination of ferritin from Dendrorhynchus zhejiangensis.

Ferritin is an important hub of iron metabolism because it stores iron during times of iron overload and releases iron during iron deficiency. Here, we present the first crystal structure of ferritin from the marine invertebrate Dendrorhynchus zhejiangensis with a 2.3 Å resolution. D. zhejiangensis ferritin (DzFer) exhibits a common cage-shaped hollow sphere with 24 subunits containing the ferroxidase centers and 3-fold and 4-fold channels. The structure of DzFer shows highly conserved catalytic residues in the ferroxidase center. The metal wire formed by ferrous ions in the 3-fold channel reveals the path that iron ions use to enter and translocate into the ferroxidase site to be oxidized and finally arrive at the nucleation site. However, the electrostatic environment of the channels and pores exhibits significant and extensive variability, suggesting that ferritins execute diverse functions in different environments.

Crystallographic characterization of ferritin from Sinonovacula constricta.

Ferritins are ubiquitous iron-binding proteins that are mainly related to iron storage, detoxification and innate immunity. Here, we present the crystal structure of a marine invertebrate ferritin from Sinonovacula constricta at a resolution of 1.98 Å. The S. constricta ferritin (ScFer) possessed some structural similarities with vertebrate ferritins, and they shared a well-conserved architecture composed of five α-helical bundles that assembled into a cage-like structure with 24-subunits. The structure of ScFer also showed iron binding sites in the 3-fold channel, ferroxidase center, and putative nucleation sites. Further, electrostatic potential calculations suggested that the electrostatic gradient of the 3-fold channel could provide a guidance mechanism for iron entering the ferritin cavity.