Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments

Abstract

22Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells 23 from oxidative damage. We have characterized the structure and function of a new member of the 24 ferritin superfamily that is sequestered within an encapsulin capsid. We show that this 25 encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent 26 manner to form a ferroxidase centre at a dimer interface. EncFtn adopts an open decameric 27 structure that is topologically distinct from other ferritins. While EncFtn acts as a ferroxidase, it 28 cannot mineralize iron. Conversely, the encapsulin shell associates with iron, but is not 29 enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for 30 iron storage. This encapsulin nanocompartment is widely distributed in bacteria and archaea and 31 represents a distinct class of iron storage system where the oxidation and mineralization of iron are 32 distributed between two proteins.

Structural characterization of the Myxococcus xanthus encapsulin and ferritin-like cargo system gives insight into its iron storage mechanism

“…Similar to classical ferritins and diiron-carboxylate proteins, both cargos contained a duplicated motif consisting of two consecutive helices and a conserved EXXH motif (Figure 5C) (Nordlund and Eklund, 1995). The diiron binding site of EncC was very similar to the previously defined binding sites of Rru_A0973 (PDB: 5DA5) and PFC_05175 (PDB: 5N5E) (Figure S7A) (He et al, 2016(He et al, , 2019. In the EncC structure one state of Fe coordination (state 1) could be observed where Fe atoms were tightly coordinated by three glutamic acid residues and a histidine residue forming a 5-coordinate Fe +2 complex (Figures 6A and S7A).…”

“…It has been proposed that the negatively charged pores located at the 5-fold axes of the shell allow the entry of positively charged Fe +2 to the lumen of the encapsulin (He et al, 2016). In that communication, Fe +2 was suggested to gain access to the metal binding sites within the central channel of the cargo protein prior to oxidation within the FOC and then released as Fe +3 (He et al, 2016). We analyzed the pores of EncA at the 2-fold, 3-fold, and 5-fold axes (Figure S9).…”

“…The function of an encapsulin is determined by the function of its cargo protein. To date, a number of different cargo proteins have been associated with encapsulins including ferritin-likeproteins (FLPs), dye-decolorizing peroxidase (DyP), iron-mineralizing encapsulin-associated firmicute (IMEF), hemerythrin, and rubrerythrin (Ahmad et al, 2011;Contreras et al, 2014;Giessen and Silver, 2017;He et al, 2016;McHugh et al, 2014;Rahmanpour and Bugg, 2013). These cargo proteins contain a short (10-40 aa) disordered peptide at their C-termini.…”

Isotope Depletion Mass Spectrometry (ID-MS) for Enhanced Top-Down Protein Fragmentation

“…We chose three well-characterised proteins as model systems for this study -encapsulated ferritin (EncFtn, 13.2 kDa), [18] carbonic anhydrase (CA, 29.3 kDa), [19] and serine palmitoyltransferase (SPT, 47.3 kDa). [20] These proteins were recombinantly expressed in E. coli using M9 minimal growth media, containing glucose and ammonium sulfate as the sole carbon and nitrogen sources.…”