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

Abstract

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.

Structural and Functional Insights into the Roles of Potential Metal-Binding Sites in Apostichopus japonicus Ferritin

“…On the basis of these results, it can be roughly concluded that the residues Asp129 and Glu132 at the three-fold channel in AjFER are probably involved in Fe 2+ capture and its translocation to the internal cavity of the protein, from where Fe 2+ reaches the ferroxidase center, similar to that proposed for BfMF [43]. Furthermore, our results confirm the ferroxidase center of AjFER indeed has a function in iron oxidation and the Glu168 residue in AjFER can act as a potential metal-ion binding site at the four-fold channel, similar to observations in previous studies [9,14]. In addition, the variants (MF, M3, and M4 proteins) exhibited lower unfolding temperatures than native AjFER, suggesting that mutations of the potential metal-binding sites in AjFER could have varying effects on its thermal stability and structural integrity.…”

“…A metal ion binding to the Cys128 residue (forming the Cd-S-Cys charge transfer complex) at the three-fold channel of ferritin revealed that metal ion coordination to sulfur can complement ionic binding during Fe 2+ transit through ion channels [43,44]. Additionally, consistent with most ferritins from marine invertebrates [7,9,14,15,45], two metal ion-binding sites (i.e., sites A and B) in the ferroxidase center were observed in the middle of four α-helical bundles per subunit in the M3 protein (Figure 4E). On the basis of the positive electron density map and the coordination environment of iron around the ferroxi-dase site, two iron ions were assigned in sites A and B of the ferroxidase center of the M3 protein.…”

“…Moreover, a previous X-ray crystal structure also indicated that the Asp129 and Glu132 residues of the three-fold channel in AjFER, corresponding to that of highly conserved residues in other marine invertebrates [13,14], indeed play a key role in iron uptake [9]. Of course, after uptake, the Fe 2+ ions are transported by a series of metal-binding residues toward a specific catalytic site, which is known as the di-iron ferroxidase center and is situated in the subunit four-helix bundle [5,9,[13][14][15]. However, although the amino acid residues located at the ferroxidase center seem to be highly conserved in most eukaryotic ferritins [7,9], the ferroxidase reaction rate may vary.…”

“…Numerous studies have demonstrated that the carboxylate side chains of Asp and Glu residues along the three-fold pores are essential for iron uptake, and these residues are involved in the translocation of Fe 2+ ions toward the ferroxidase center for rapid catalytic activity in human H-ferritin (HuHF) and frog M-ferritin [6,12]. Moreover, a previous X-ray crystal structure also indicated that the Asp129 and Glu132 residues of the three-fold channel in AjFER, corresponding to that of highly conserved residues in other marine invertebrates [13,14], indeed play a key role in iron uptake [9]. Of course, after uptake, the Fe 2+ ions are transported by a series of metal-binding residues toward a specific catalytic site, which is known as the di-iron ferroxidase center and is situated in the subunit four-helix bundle [5,9,[13][14][15].…”

“…For instance, De Meulenaere et al found that the ferroxidase velocity of the marine invertebrate Chaetopterus ferritin (ChF) was up to eight times faster than that of HuHF, which is known as a conventionally accepted representative ferritin [15]. Furthermore, recent studies have suggested that the Glu168 residue at the four-fold channel can act as a potential binding site of metal ions in some marine invertebrate ferritins [9,14,15], but there is still a lack of sufficient evidence to determine whether this metal-binding site is related to the catalytic function of ferritin. Accordingly, some unique functions of marine invertebrate ferritins have not been observed.…”