Conservation of the structural and functional architecture of encapsulated ferritins in bacteria and archaea

Abstract

+44(0)191 208 4855, Jon.marles-wright1@ncl.ac.uk; David J. Clarke, +44(0)131 650 4808, Dave.clarke@ed.ac.uk. ABSTRACT Iron is an essential element for many biological processes; however, due to its high reactivity iron can also be very toxic, producing reactive oxygen species through Fenton chemistry. Ferritins protect the cell from oxidative stress by catalytically converting Fe(II) into less toxic Fe(III) and storing the resulting iron minerals within their core. Encapsulated ferritins (EncFtn) are a sub-family of ferritin-like proteins, which are widely distributed in all bacterial and archaeal phyla. We recently characterised the Rhodospirillum rubrum EncFtn, showing that although enzymatically active, due to its open structure it requires the association with an encapsulin nanocage in order to act as an iron store. Given the wide distribution of the EncFtn family in organisms with diverse environmental niches, a question arises as to whether the structure and catalytic activity is conserved across the family. Here we structurally characterise two EncFtn members from the halophile Haliangium ochraceum and the thermophile Pyrococcus furiosus, which show the same distinct annular decamer topology observed in R. rubrum EncFtn, with the ferroxidase centre (FOC) formed between one of the dimer interfaces. Solution and native mass

Dissecting the structural and functional roles of a putative metal entry site in encapsulated ferritins

“…The E34A variant had significantly more protein in the 82 ml peak when compared to the EncFtn-WT and E31A variant, suggesting that this change affects the stability of the protein complex. Based on our prior work, these peaks correspond to decamer, tetramer, and dimer species, respectively (14,24).…”

“…To interrogate the stability and oligomeric states of the EncFtn variants, we performed native MS and ion mobility MS experiments on samples purified by size-exclusion chromatography ( Figure 5) (14). In our initial studies we found that EncFtn-W38A was not amenable to native mass spectrometry analysis due to poor solubility in native MS ammonium acetate buffer; consequently, native MS was performed on EncFtn-WT, EncFtn-E31A, EncFtn-E34A, and EncFtn-W38G.…”

“…In our previous work we identified a number of conserved secondary metal-binding sites in EncFtn proteins (14,15). A partially occupied metal ion site was present on the outer surface (proposed exit site) of the EncFtn decamer, and a highly occupied metal ion site was present on the inner face (proposed entry site), which is proximal to the ferroxidase center (Figure 2A and B).…”

“…The ferritin superfamily consists of a number of structurally and functionally distinct members, each of which is built around a fourhelix bundle scaffold which forms an ironbinding di-iron ferroxidase active site (the ferroxidase center, FOC) (1). The quaternary structure of these protein families shows a high degree of variation: from monomeric rubrerythrin proteins (2,3); dimeric ironmineralizing encapsulin-associated firmicute (IMEF) proteins (4); dodecameric DNA Protection in Starved (DPS) cells proteins and DPS-like (DPSL) proteins (5)(6)(7)(8)(9); the 24-meric classical ferritins (Ftn) and bacterioferritins (Bfr) (10)(11)(12)(13); and the decameric encapsulated ferritins (EncFtn) (14,15). The rubrerythrin and DPS proteins play key roles in the protection of cells from oxidative stress, while DPS and the other members of the ferritin superfamily are vital iron stores (7,(16)(17)(18)(19)(20)(21)(22).…”

“…The nanocages formed by the quaternary structure of DPS, DPSL, Ftn, and Bfr allow iron oxidation and mineralization to occur within a biochemically privileged environment within the cell, which protects the host cell from ironinduced oxidative stress. Interestingly, the recently described IMEF and EncFtn proteins both have ferroxidase activity, but they do not form nanocage structures on their own, instead they must be localized to the lumen of encapsulin nanocages to form a competent iron-store (4,14,15). EncFtn and IMEF are directed into encapsulins by a terminal localization sequence that binds to the interior wall of encapsulin.…”