Human calprotectin is an iron-sequestering host-defense protein

Abstract

Human calprotectin (CP) is a metal-chelating antimicrobial protein of the innate immune response. The current working model states that CP sequesters manganese and zinc from pathogens. We report the discovery that CP chelates iron and deprives bacteria of this essential nutrient. Elemental analysis of CP-treated growth medium establishes that CP reduces the concentrations of manganese, iron and zinc. Microbial growth studies reveal that iron depletion by CP contributes to the growth inhibition of bacterial pathogens. Biochemical investigations demonstrate that CP coordinates Fe(II) at an unusual hexahistidine motif, and the Mössbauer spectrum of 57Fe(II)-bound CP is consistent with coordination of high-spin Fe(II) at this site (δ = 1.20 mm/s, ΔEQ = 1.78 mm/s). In the presence of Ca(II), CP turns on its iron-sequestering function and exhibits subpicomolar affinity for Fe(II). Our findings expand the biological coordination chemistry of iron and support a previously unappreciated role for CP in mammalian iron homeostasis.

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Figure 1: CP houses two transition metal–binding sites at the S100A8–S100A9 interface.
Figure 2: CP depletes metals from bacterial growth medium.
Figure 3: Metal supplementation growth studies.
Figure 4: The antimicrobial activity of CP against E. coli, S. aureus and L. plantarum.
Figure 5: Inhibition of bacterial iron acquisition by CP.
Figure 6: Hexahistidine Fe(II) coordination by CP.
Figure 7: CP binds Fe(II) with remarkably high affinity in a Ca(II)-dependent manner.

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Acknowledgements

Research on Fe(II)-CP in the Nolan Laboratory was supported by the Office of the Director of the US National Institutes of Health (NIH grant 1DP2OD007045, E.M.N.), the MIT Center for Environmental Health Sciences (NIH P30-ES002109, E.M.N.), the Sloan Foundation and the Kinship Foundation (Searle Scholar Award, E.M.N.). T.G.N. is a recipient of the NSF Graduate Research Fellowship. Any opinion, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the NSF. We thank R. Laufhutte for performing the ICP-MS and ICP-OES analyses, A.J. Wommack for synthesizing ZP1 and J. Stubbe and members of her laboratory for guidance on the 55Fe experiments and for providing the facilities to work with radioactivity. We acknowledge the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for providing the S. aureus USA300 JE2 parent strain of the Nebraska Transposon Mutant Library (NTML) that is supported by NIH NIAID grant HHSN272200700055C.

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T.G.N. and E.M.N. designed the research. T.G.N. prepared the ICP-MS, ICP-OES and Mössbauer spectroscopy samples and conducted the microbial growth assays, 55Fe uptake studies, analytical SEC, ZP1 Kd,Fe(II) determination and metal-ion competition experiments. B.Z. and C.K. performed and analyzed the Mössbauer spectroscopy. T.G.N. and E.M.N. analyzed the results and wrote the paper.

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Correspondence to Elizabeth M Nolan.

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Supplementary Results, Supplementary Tables 1–14, Supplementary Figures 1–23 and Supplementary Note. (PDF 4585 kb)

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Nakashige, T., Zhang, B., Krebs, C. et al. Human calprotectin is an iron-sequestering host-defense protein. Nat Chem Biol 11, 765–771 (2015). https://doi.org/10.1038/nchembio.1891

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