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 (57)Fe(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.

Figure 1. CP houses two transition-metal binding sites at the S100A8/S100A9 interface

(a) CP heterodimer from the structure of the Mn(II)-, Ca(II)-, and Na(I)-bound heterotetramer (PDB: 4XJK). S100A8 is shown in green. S100A9 is shown in blue. The calcium ions are shown as yellow spheres. The sodium ions are shown as purple spheres. The manganese ion is shown as a magenta sphere. The amino acid residues at the metal binding sites are shown as orange sticks. (b) The His₃Asp motif (site 1) is comprised of (A8)His83, (A8)His87, (A9)His20, and (A9)Asp30. (c) The His₆ motif (site 2) is comprised of (A8)His17, (A8)His27, (A9)His91, (A9)His95, (A9)His103, and (A9)His105.

Figure 2. CP depleted metals from bacterial growth medium

Metal analysis of Mn (a), Zn (b), and Fe (c) concentrations in CP-treated and untreated growth medium (250 µg/mL CP, ~3 mM BME) in the absence (white bars) and presence (gray bars) of a 2-mM Ca(II) supplement (mean ± SEM, n ≥ 3).

Figure 3. Metal supplementation growth studies

Growth curves of Gram-negative bacteria Acinetobacter baumannii (a), laboratory and enterohemorrhagic strains of Escherichia coli (b,c), Klebsiella pneumoniae (d), and Pseudomonas aeruginosa (e), and Gram-positive bacteria Bacillus cereus (f) and Staphylococcus aureus (g,h) cultured in medium (+Ca(II), +BME) treated with CP-Ser (250 µg/mL) and supplemented with 0.15 µM Mn(II), 3 µM Fe(II), and/or 5 µM Zn(II) (mean ± SEM, n ≥ 3).

Figure 4. The antimicrobial activity of CP against E. coli, S. aureus, and L. plantarum

(a,b) Antibacterial activity of CP-Ser, ΔHis₃Asp, and ΔHis₄ preincubated with 0.9 equiv of Fe(II) against E. coli and S. aureus (t = 20 h, mean ± SEM, n = 6). (c,d) Antibacterial activity of CP against L. plantarum in the absence and presence of 2-mM Ca(II) and 3-mM BME supplements (t = 20 h, mean ± SEM, n = 3). (e) Growth of L. plantarum in medium treated with CP-Ser (500 µg/mL) supplemented with 10 µM Mn(II) and 10 µM Zn(II) (mean ± SEM, n = 3).

Figure 5. Inhibition of bacterial iron acquisition by CP

Uptake of radiolabeled ⁵⁵Fe by E. coli (a) and P. aeruginosa (b) treated with 500 µg/mL CP-Ser (squares) or ΔΔ (diamonds) compared with untreated bacteria (circles). The upper panels show the amount of ⁵⁵Fe uptake per mL of bacterial culture over the 2-h experiment. The lower panels indicate the corresponding OD₆₀₀ values of the cultures at each time point. The mean ± SEM values for ⁵⁵Fe and OD₆₀₀ are shown (n = 4).

Figure 6. Hexahistidine Fe(II) coordination by CP

(a–c) Analytical SEC chromatograms of CP-Ser (400 µM) incubated with no metal, 5 equiv of Fe(II), and 5 equiv of Fe(III), and quantification of Fe and CP in eluent fractions. (d–f) Analytical SEC chromatograms of CP (20 µM) incubated with 10 equiv of Fe(II). Chromatograms for CP αβ (no metal) and α₂β₂ (+Ca(II)) are provided for reference. Peak intensities are normalized to 1. (g) The 4.2-K/53-mT Mössbauer spectrum of ⁵⁷Fe(II)-bound CP-Ser (black vertical bars). Simulation using the parameters described in the main text is shown as the blue line, and the Mössbauer spectrum of Fe(II) sulfate in 50 mM Tris, pH 7.5 recorded under the same conditions is shown as the red line.

Figure 7. CP binds Fe(II) with remarkably high affinity in a Ca(II)-dependent manner

(a) ZP1 (1 µM) emission response to 3.5 µM Fe(II) in the absence (circles) and presence (squares) of 4 µM CP-Ser with addition of Ca(II). The red markers represent the 200-µM Ca(II) samples spiked with 4 µM Fe(II) (mean, n = 2). (b) ZP1 (1 µM) emission response to Fe(II) in the presence of 4 µM CP with or without 50 equiv of Ca(II) (mean ± SDM, n = 3). (c) Room-temperature EPR spectra of Mn(II) (black), CP-Ser in the presence of 50 equiv of Ca(II) and 0.9 equiv of Mn(II) (red), and CP-Ser in the presence of 50 equiv of Ca(II) and 0.9 equiv of Mn(II) with the addition of 0.9 equiv of Fe(II) (blue). (d) The concentration of free Mn(II) determined by room-temperature EPR (mean ± SDM, n = 3). (e) The concentration of unbound Fe(II) in ΔHis₃Asp samples containing 0.9 equiv of Fe(II) after addition of 0.9 equiv of Zn(II) with and without 50 equiv of Ca(II) (mean ± SDM, n = 3). Control samples containing 25 µM protein, 0.9 equiv of Fe(II), and/or 50 equiv of Ca(II) are described in the right panel.