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

Abstract

Encapsulated ferritins belong to the universally distributed ferritin superfamily, which function as iron detoxification and storage systems. Encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store that is capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question, due to differences in organization of the ferroxidase catalytic site and neighboring secondary metal binding sites. We have previously identified a putative metal binding site on the inner surface of the Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase center. Here we present a comprehensive structural and functional study to investigate the functional relevance of this putative iron entry site by means of enzymatic assays, mass-spectrometry, and X-ray crystallography. We show that catalysis occurs in the ferroxidase center and suggest a dual role for the secondary site, which both serves to attract metal ions to the ferroxidase center and acts as a flow-restricting valve to limit the activity of the ferroxidase center. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, while enhancing the ability of the encapsulated ferritin to undergo catalysis, does not influence the function of the secondary site. Our study demonstrates a novel molecular mechanism by which substrate flux to the ferroxidase center is controlled, potentially to ensure that iron oxidation is productively coupled to mineralization.

Cryo-EM structure of a thermostable bacterial nanocompartment

“…Nonetheless, the bottom of the fivefold axis channel consists of the hydrophobic sides of tyrosine side chains, suggesting that iron may not be able to freely diffuse through. This is consistent with a report that the encapsulated ferritin-like protein has an entry site that prevents rapid passage to the ferroxidase center (Piergentili et al, 2020). Iron transport may be regulated such that iron ions are conducted along a defined pathway in TmEnc towards the ferritin-like protein, as opposed to random diffusion.…”

Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment

“…Furthermore, both the engaged and unengaged localization sequences present a 'soft' steric barrier to the diffusion of substrates. It is notable that the ferroxidase activity of the Enc:EncFtn complex is significantly higher than the isolated EncFtn protein (36). The dynamic behavior of the encapsulin five-fold pores, which is apparent in our 3DVA movies, may increase EncFtn activity by enhancing diffusion of substrates into the lumen of the nanocompartment.…”

“…Our data were collected on apo-Enc:EncFtn complexes, and thus is it not possible to infer the nature of the iron mineralization pathway within the encapsulin nanocage. Further, careful work must be performed to titrate iron into the complex prior to structural analysis to gain insight into the flow of metal ions from the exterior to the interior of the encapsulin and to determine if metalation influences the conformational flexibility of the EncFtn within the encapsulin nanocage, as we have demonstrated for isolated EncFtn proteins (He et al, 2019(He et al, , 2016Piergentili et al, 2020). Finally, the nature of the iron mineral and its localization within the encapsulin nanocage is still to be determined.…”

“…The enzymatic activity of Empty-Enc and Loaded-Enc were assessed by ferroxidase assay, as previously described (Piergentili, 2020). Fe(II) samples were prepared by dissolving FeSO4.7H2O in HCl 0.1 % (v/v) under anaerobic conditions.…”

Recent advances in the structural biology of encapsulin bacterial nanocompartments

“…Several structures of cargo proteins are also available ( Fig. 1 B) ( Giessen et al, 2019 , He et al, 2019 , Piergentili et al, 2020 , Tang et al, 2021 , Xiong et al, 2020 ).…”