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Structural basis of haem-iron acquisition by fungal pathogens

Abstract

Pathogenic microorganisms must cope with extremely low free-iron concentrations in the host's tissues. Some fungal pathogens rely on secreted haemophores that belong to the Common in Fungal Extracellular Membrane (CFEM) protein family, to extract haem from haemoglobin and to transfer it to the cell's interior, where it can serve as a source of iron. Here we report the first three-dimensional structure of a CFEM protein, the haemophore Csa2 secreted by Candida albicans. The CFEM domain adopts a novel helical-basket fold that consists of six α-helices, and is uniquely stabilized by four disulfide bonds formed by its eight signature cysteines. The planar haem molecule is bound between a flat hydrophobic platform located on top of the helical basket and a peripheral N-terminal ‘handle’ extension. Exceptionally, an aspartic residue serves as the CFEM axial ligand, and so confers coordination of Fe3+ haem, but not of Fe2+ haem. Histidine substitution mutants of this conserved Asp acquired Fe2+ haem binding and retained the capacity to extract haem from haemoglobin. However, His-substituted CFEM proteins were not functional in vivo and showed disturbed haem exchange in vitro, which suggests a role for the oxidation-state-specific Asp coordination in haem acquisition by CFEM proteins.

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Figure 1: C. albicans Csa2 is a secreted haemophore.
Figure 2: Csa2 and rCsa2 exchange haem with Rbt5 and Pga7.
Figure 3: The crystal structure of the Csa2 CFEM protein reveals a novel helical-basket fold.
Figure 4: CFEM has a unique binding site for haem and an exceptional iron coordination.
Figure 5: Histidine-substituted CFEM mutants bind haem and extract haem from haemoglobin, but are inactive and defective in haem exchange.
Figure 6: Schematic pathway of haem-iron acquisition in Candidaceae.

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Acknowledgements

We are grateful to D. Hiya (Technion Center for Structural Biology) for help with protein crystallization, T. Ziv (Smoler Proteomics Center) for mass spectroscopy analysis, G. Kuznets and Y. Gorelik for constructing plasmids KB2366 and KB2221/2, U. Roy for producing Csa2 cysteine mutants, M. Lebendiker (Hebrew University of Jerusalem) for advice with SEC-MALS, R. Zarivach (Ben-Gurion University) for the Origami B strain and O. Lewinson, I. Silman, N. Adir, N. Levanon, A. Haber and S. Selig for discussions and critical reading of the manuscript. This research was supported by grants from the Israel Science Foundation and the Ministry of Health's Chief Scientist Office to D.K. H.D. thanks the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement No. 330879-MC-CHOLESTRUCTURE for financial support.

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L.N., Z.W., M.P. and D.K. constructed the plasmids and strains, purified the proteins and performed experiments, H.A. performed the SEC-MALS analysis, H.D. crystallized the protein and determined its structure, and H.D. and D.K. developed the project, interpreted the data and wrote the paper.

Corresponding authors

Correspondence to Hay Dvir or Daniel Kornitzer.

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Supplementary Figures 1–9, Supplementary Tables 1–3, Supplementary References (PDF 4321 kb)

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Nasser, L., Weissman, Z., Pinsky, M. et al. Structural basis of haem-iron acquisition by fungal pathogens. Nat Microbiol 1, 16156 (2016). https://doi.org/10.1038/nmicrobiol.2016.156

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