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

Abstract

Ferritins are a large family of intracellular proteins that protect the cell from oxidative stress by catalytically converting Fe(II) into less toxic Fe(III) and storing 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. The recently characterized Rhodospirillum rubrum EncFtn displays an unusual structure when compared with classical ferritins, with an open decameric structure that is enzymatically active, but unable to store iron. This EncFtn must be associated 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 this unusual structure is conserved across the family. Here, we characterize EncFtn proteins from the halophile Haliangium ochraceum and the thermophile Pyrococcus furiosus, which show the conserved annular pentamer of dimers topology. Key structural differences are apparent between the homologues, particularly in the centre of the ring and the secondary metal-binding site, which is not conserved across the homologues. Solution and native mass spectrometry analyses highlight that the stability of the protein quaternary structure differs between EncFtn proteins from different species. The ferroxidase activity of EncFtn proteins was confirmed, and we show that while the quaternary structure around the ferroxidase centre is distinct from classical ferritins, the ferroxidase activity is still inhibited by Zn(II). Our results highlight the common structural organization and activity of EncFtn proteins, despite diverse host environments and contexts within encapsulins.

Mass spectrometry reveals the assembly pathway of encapsulated ferritins and highlights a dynamic ferroxidase interface

“…3 In addition, prior native MS studies have also revealed that the EncFtn from the halophile Haliangium Ochraceum exists as a dynamic structure in multiple even-numbered oligomerisation states (dimer, tetramer, hexamer and decamer). 9 This observation implies that higher order oligomers are formed by the association of dimer units; presumably either via FOC dimer association, or non-FOC dimer association ( Fig. 1D and E).…”

“…As previously reported, native MS analysis of Hoch-WT reveals a series of oligomerisation states. 9 The major species is dimer (+9 to +11) ( Fig. 2A, green circles), with some tetramer (13+ to 15+), hexamer (17+ to 18+) and decamer (22+ to 25+) species also observed ( Fig.…”

“…3,9 This is formed from a pentamer of dimers, the topology of which results in two distinct dimer interfaces. One dimer is structurally similar to the four helix-bundle of the classical ferritin monomer 9 and is formed by the two main helices of two EncFtn monomers. This dimer contains the di-iron ferroxidase centre (FOC) active site and is known as the FOC dimer ( Fig.…”

Large-scale computational discovery and analysis of virus-derived microbial nanocompartments

“…S2). 14,30 Unlike ferritin cages with higher symmetries, Flp cargo proteins cannot store precipitated iron in a soluble form by themselves and rely on the encapsulin shell to achieve iron precipitate sequestration. Similar to the ubiquitous ferritin iron storage cages, Flp encapsulin systems might play a dual role in oxidative stress resistance and iron homeostasis.…”

Mass Spectrometry Reveals the Assembly Pathway of Encapsulated Ferritins and Highlights a Dynamic Ferroxidase Interface

“…1A). 3,9 This is formed from a pentamer of dimers, the topology of which results in two distinct dimer interfaces. One dimer is structurally similar to the four helix-bundle of the classical ferritin monomer 9 and is formed by the two main helices of two EncFtn monomers.…”