A fusion of the Bacteroides fragilis ferrous iron import proteins reveals a role for FeoA in stabilizing GTP-bound FeoB.

Autor:	Sestok AE; Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, Maryland, USA., Brown JB; Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, Maryland, USA., Obi JO; Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland, USA., O'Sullivan SM; Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, Maryland, USA., Garcin ED; Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, Maryland, USA; Laboratoire d'Information Génomique et Structurale, UMR7256, Aix-Marseille Université, Campus de Luminy, Marseille, France., Deredge DJ; Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland, USA., Smith AT; Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, Maryland, USA. Electronic address: smitha@umbc.edu.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2022 Apr; Vol. 298 (4), pp. 101808. Date of Electronic Publication: 2022 Mar 08.
DOI:	10.1016/j.jbc.2022.101808
Abstrakt:	Iron is an essential element for nearly all organisms, and under anoxic and/or reducing conditions, Fe 2+ is the dominant form of iron available to bacteria. The ferrous iron transport (Feo) system is the primary prokaryotic Fe 2+ import machinery, and two constituent proteins (FeoA and FeoB) are conserved across most bacterial species. However, how FeoA and FeoB function relative to one another remains enigmatic. In this work, we explored the distribution of feoAB operons encoding a fusion of FeoA tethered to the N-terminal, G-protein domain of FeoB via a connecting linker region. We hypothesized that this fusion poises FeoA to interact with FeoB to affect function. To test this hypothesis, we characterized the soluble NFeoAB fusion protein from Bacteroides fragilis, a commensal organism implicated in drug-resistant infections. Using X-ray crystallography, we determined the 1.50-Å resolution structure of BfFeoA, which adopts an SH3-like fold implicated in protein-protein interactions. Using a combination of structural modeling, small-angle X-ray scattering, and hydrogen-deuterium exchange mass spectrometry, we show that FeoA and NFeoB interact in a nucleotide-dependent manner, and we mapped the protein-protein interaction interface. Finally, using guanosine triphosphate (GTP) hydrolysis assays, we demonstrate that BfNFeoAB exhibits one of the slowest known rates of Feo-mediated GTP hydrolysis that is not potassium-stimulated. Importantly, truncation of FeoA from this fusion demonstrates that FeoA-NFeoB interactions function to stabilize the GTP-bound form of FeoB. Taken together, our work reveals a role for FeoA function in the fused FeoAB system and suggests a function for FeoA among prokaryotes. Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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