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

Lookup NU author(s): Dr Cecilia Piergentili, Dr Will Stanley, Laurene Adam, Dr Arnaud Basle, Dr Kevin Waldron, Dr Jon Marles-Wright

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This is the authors' accepted manuscript of an article that has been published in its final definitive form by American Society for Biochemistry and Molecular Biology, 2020.

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Abstract

Encapsulated ferritins belong to the universally distributed ferritin superfamily, whose members 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 because of the 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, MS, 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, although 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.


Publication metadata

Author(s): Piergentili C, Ross J, He D, Gallagher KJ, Stanley WA, Adam L, Mackay CL, Baslé A, Waldron KJ, Clarke DJ, Marles-Wright J

Publication type: Article

Publication status: Published

Journal: Journal of Biological Chemistry

Year: 2020

Volume: 295

Pages: 15511-15526

Print publication date: 13/11/2020

Online publication date: 02/09/2020

Acceptance date: 02/09/2020

Date deposited: 13/11/2020

ISSN (print): 0021-9258

ISSN (electronic): 1083-351X

Publisher: American Society for Biochemistry and Molecular Biology

URL: https://doi.org/10.1074/jbc.RA120.014502

DOI: 10.1074/jbc.RA120.014502


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