Human calprotectin is an iron-sequestering host-defense protein


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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  1. 1

    Bertini, I., Gray, H.B., Stiefel, E.I. & Valentine, J.S. Biological Inorganic Chemistry: Structure and Reactivity (University Science Books, 2007).

  2. 2

    Weinberg, E.D. Nutritional immunity: host's attempt to withhold iron from microbial invaders. J. Am. Med. Assoc. 231, 39–41 (1975).

    CAS  Article  Google Scholar 

  3. 3

    Fischbach, M.A., Lin, H., Liu, D.R. & Walsh, C.T. How pathogenic bacteria evade mammalian sabotage in the battle for iron. Nat. Chem. Biol. 2, 132–138 (2006).

    CAS  Article  Google Scholar 

  4. 4

    Hood, M.I. & Skaar, E.P. Nutritional immunity: transition metals at the pathogen-host interface. Nat. Rev. Microbiol. 10, 525–537 (2012).

    CAS  Article  Google Scholar 

  5. 5

    Cassat, J.E. & Skaar, E.P. Iron in infection and immunity. Cell Host Microbe 13, 509–519 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6

    Diaz-Ochoa, V.E., Jellbauer, S., Klaus, S. & Raffatellu, M. Transition metal ions at the crossroads of mucosal immunity and microbial pathogenesis. Front. Cell. Infect. Microbiol. 4, 2 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  7. 7

    Sohnle, P.G., Collins-Lech, C. & Wiessner, J.H. The zinc-reversible antimicrobial activity of neutrophil lysates and abscess fluid supernatants. J. Infect. Dis. 164, 137–142 (1991).

    CAS  Article  Google Scholar 

  8. 8

    Corbin, B.D. et al. Metal chelation and inhibition of bacterial growth in tissue abscesses. Science 319, 962–965 (2008).

    CAS  Article  Google Scholar 

  9. 9

    Kehl-Fie, T.E. et al. Nutrient metal sequestration by calprotectin inhibits bacterial superoxide defense, enhancing neutrophil killing of Staphylococcus aureus. Cell Host Microbe 10, 158–164 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10

    Brophy, M.B., Hayden, J.A. & Nolan, E.M. Calcium ion gradients modulate the zinc affinity and antibacterial activity of human calprotectin. J. Am. Chem. Soc. 134, 18089–18100 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11

    Hayden, J.A., Brophy, M.B., Cunden, L.S. & Nolan, E.M. High-affinity manganese coordination by human calprotectin is calcium dependent and requires the histidine-rich site formed at the dimer interface. J. Am. Chem. Soc. 135, 775–787 (2013).

    CAS  Article  Google Scholar 

  12. 12

    Damo, S.M. et al. Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens. Proc. Natl. Acad. Sci. USA 110, 3841–3846 (2013).

    CAS  Article  Google Scholar 

  13. 13

    Brophy, M.B., Nakashige, T.G., Gaillard, A. & Nolan, E.M. Contributions of the S100A9 C-terminal tail to high-affinity Mn(II) chelation by the host-defense protein human calprotectin. J. Am. Chem. Soc. 135, 17804–17817 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  14. 14

    Vogl, T., Leukert, N., Barczyk, K., Strupat, K. & Roth, J. Biophysical characterization of S100A8 and S100A9 in the absence and presence of bivalent cations. Biochim. Biophys. Acta 1763, 1298–1306 (2006).

    CAS  Article  Google Scholar 

  15. 15

    Gagnon, D.M. et al. Manganese-binding properties of human calprotectin under conditions of high and low calcium: X-ray crystallographic and advanced electron paramagnetic resonance spectroscopic analysis. J. Am. Chem. Soc. 137, 3004–3016 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16

    Korndörfer, I.P., Brueckner, F. & Skerra, A. The crystal structure of the human (S100A8/S100A9)2 heterotetramer, calprotectin, illustrates how conformational changes of interacting α-helices can determine specific association of two EF-hand proteins. J. Mol. Biol. 370, 887–898 (2007).

    Article  Google Scholar 

  17. 17

    Zaharik, M.L. & Finlay, B.B. Mn2+ and bacterial pathogenesis. Front. Biosci. 9, 1035–1042 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18

    Papp-Wallace, K.M. & Maguire, M.E. Manganese transport and the role of manganese in virulence. Annu. Rev. Microbiol. 60, 187–209 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19

    Jacobsen, F.E., Kazmierczak, K.M., Lisher, J.P., Winkler, M.E. & Giedroc, D.P. Interplay between manganese and zinc homeostasis in the human pathogen Streptococcus pneumoniae. Metallomics 3, 38–41 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20

    Aguirre, J.D. et al. A manganese-rich environment supports superoxide dismutase activity in a Lyme disease pathogen, Borrelia burgdorferi. J. Biol. Chem. 288, 8468–8478 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21

    Lisher, J.P. & Giedroc, D.P. Manganese acquisition and homeostasis at the host-pathogen interface. Front. Cell. Infect. Microbiol. 3, 91 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  22. 22

    Irving, H. & Williams, R.J.P. The stability of transition-metal complexes. J. Chem. Soc. 3192–3210 (1953).

  23. 23

    Brophy, M.B. & Nolan, E.M. Manganese and microbial pathogenesis: sequestration by the mammalian immune system and utilization by microorganisms. ACS Chem. Biol. 10, 641–651 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24

    Archibald, F.S. & Fridovich, I. Manganese and defenses against oxygen toxicity in Lactobacillus plantarum. J. Bacteriol. 145, 442–451 (1981).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Frey, P.A. & Reed, G.H. The ubiquity of iron. ACS Chem. Biol. 7, 1477–1481 (2012).

    CAS  Article  Google Scholar 

  26. 26

    Münck, E. in Physical Methods in Bioinorganic Chemistry: Spectroscopy and Magnetism (ed. Que, L., Jr.) 287–319 (University Science Books, 2000).

  27. 27

    Greenwood, N.N. & Gibb, T.C. Mössbauer Spectroscopy. (Springer, 1971).

  28. 28

    Asch, L., Adloff, J.P., Friedt, J.M. & Danon, J. Motional effects in the Mössbauer spectra of iron(II) hexammines. Chem. Phys. Lett. 5, 105–108 (1970).

    CAS  Article  Google Scholar 

  29. 29

    Bossek, U. et al. Exchange coupling in an isostructural series of face-sharing bioctahedral complexes [LMII(μ-X)3M(II)L]BPh4 (M = Mn, Fe, Co, Ni, Zn; X = Cl, Br; L=1,4,7-trimethyl-1,4,7-triazacyclononane). Inorg. Chem. 36, 2834–2843 (1997).

    CAS  Article  Google Scholar 

  30. 30

    Cotruvo, J.A. Jr. & Stubbe, J. Metallation and mismetallation of iron and manganese proteins in vitro and in vivo: the class I ribonucleotide reductases as a case study. Metallomics 4, 1020–1036 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31

    Walkup, G.K., Burdette, S.C., Lippard, S.J. & Tsien, R.Y. A new cell-permeable fluorescent probe for Zn2+. J. Am. Chem. Soc. 122, 5644–5645 (2000).

    CAS  Article  Google Scholar 

  32. 32

    Gibbs, C.R. Characterization and application of ferrozine iron reagent as a ferrous iron indicator. Anal. Chem. 48, 1197–1201 (1976).

    CAS  Article  Google Scholar 

  33. 33

    Stookey, L.L. Ferrozine—a new spectrophotometric reagent for iron. Anal. Chem. 42, 779–781 (1970).

    CAS  Article  Google Scholar 

  34. 34

    Moroz, O.V., Blagova, E.V., Wilkinson, A.J., Wilson, K.S. & Bronstein, I.B. The crystal structures of human S100A12 in apo form and in complex with zinc: new insights into S100A12 oligomerization. J. Mol. Biol. 391, 536–551 (2009).

    CAS  Article  Google Scholar 

  35. 35

    Brodersen, D.E., Nyborg, J. & Kjeldgaard, M. Zinc-binding site of an S100 protein revealed. Two crystal structures of Ca2+-bound human psoriasin (S100A7) in the Zn2+-loaded and Zn2+-free states. Biochemistry 38, 1695–1704 (1999).

    CAS  Article  Google Scholar 

  36. 36

    Ostendorp, T., Diez, J., Heizmann, C.W. & Fritz, G. The crystal structures of human S100B in the zinc- and calcium-loaded state at three pH values reveal zinc ligand swapping. Biochim. Biophys. Acta 1813, 1083–1091 (2011).

    CAS  Article  Google Scholar 

  37. 37

    Liu, J.Z. et al. Zinc sequestration by the neutrophil protein calprotectin enhances Salmonella growth in the inflamed gut. Cell Host Microbe 11, 227–239 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38

    Königsberger, L.C., Königsberger, E., May, P.M. & Hefter, G.T. Complexation of iron(III) and iron(II) by citrate. Implications for iron speciation in blood plasma. J. Inorg. Biochem. 78, 175–184 (2000).

    Article  Google Scholar 

  39. 39

    Mills, S.A. & Marletta, M.A. Metal-binding characteristics and role of iron oxidation in the ferric uptake regulator from Escherichia coli. Biochemistry 44, 13553–13559 (2005).

    CAS  Article  Google Scholar 

  40. 40

    Mizuno, K., Whittaker, M.M., Bächinger, H.P. & Whittaker, J.W. Calorimetric studies on the tight binding metal interactions of Escherichia coli manganese superoxide dismutase. J. Biol. Chem. 279, 27339–27344 (2004).

    CAS  Article  Google Scholar 

  41. 41

    Carver, G., Tregenna-Piggott, P.L.W., Barra, A.-L., Neels, A. & Stride, J.A. Spectroscopic and structural characterization of the [Fe(imidazole)6]2+ cation. Inorg. Chem. 42, 5771–5777 (2003).

    CAS  Article  Google Scholar 

  42. 42

    Nistor, A., Shova, S., Cazacu, M. & Lazar, A. Hexakis(1H-imidazole-κN3)iron(II) sulfate–1H-imidazole (1/2). Acta Crystallogr. Sect. E Struct. Rep. Online 67, m1600–m1601 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43

    Singh, P.K., Parsek, M.R., Greenberg, E.P. & Welsh, M.J. A component of innate immunity prevents bacterial biofilm development. Nature 417, 552–555 (2002).

    CAS  Article  Google Scholar 

  44. 44

    Flo, T.H. et al. Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature 432, 917–921 (2004).

    CAS  Article  Google Scholar 

  45. 45

    Cartron, M.L., Maddocks, S., Gillingham, P., Craven, C.J. & Andrews, S.C. Feo –transport of ferrous iron into bacteria. Biometals 19, 143–157 (2006).

    CAS  Article  Google Scholar 

  46. 46

    Stojiljkovic, I., Cobeljic, M. & Hantke, K. Escherichia coli K-12 ferrous iron–uptake mutants are impaired in their ability to colonize the mouse intestine. FEMS Microbiol. Lett. 108, 111–115 (1993).

    CAS  Article  Google Scholar 

  47. 47

    Konings, A.F. et al. Pseudomonas aeruginosa uses multiple pathways to acquire iron during chronic infection in cystic fibrosis lungs. Infect. Immun. 81, 2697–2704 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48

    Velayudhan, J. et al. Iron acquisition and virulence in Helicobacter pylori: a major role for FeoB, a high-affinity ferrous iron transporter. Mol. Microbiol. 37, 274–286 (2000).

    CAS  Article  Google Scholar 

  49. 49

    Carter, P. Spectrophotometric determination of serum iron at the submicrogram level with a new reagent (ferrozine). Anal. Biochem. 40, 450–458 (1971).

    CAS  Article  Google Scholar 

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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).

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