Calcium ion gradients modulate the zinc affinity and antibacterial activity of human calprotectin

Abstract

Calprotectin (CP) is an antimicrobial protein produced and released by neutrophils that inhibits the growth of pathogenic microorganisms by sequestering essential metal nutrients in the extracellular space. In this work, spectroscopic and thermodynamic metal-binding studies are presented to delineate the zinc-binding properties of CP. Unique optical absorption and EPR spectroscopic signatures for the interfacial His(3)Asp and His(4) sites of human calprotectin are identified by using Co(II) as a spectroscopic probe. Zinc competition titrations employing chromophoric Zn(II) indicators provide a 2:1 Zn(II):CP stoichiometry, confirm that the His(3)Asp and His(4) sites of CP coordinate Zn(II), and reveal that the Zn(II) affinity of both sites is calcium-dependent. The calcium-insensitive Zn(II) competitor ZP4 affords dissociation constants of K(d1) = 133 ± 58 pM and K(d2) = 185 ± 219 nM for CP in the absence of Ca(II). These values decrease to K(d1) ≤ 10 pM and K(d2) ≤ 240 pM in the presence of excess Ca(II). The K(d1) and K(d2) values are assigned to the His(3)Asp and His(4) sites, respectively. In vitro antibacterial activity assays indicate that the metal-binding sites and Ca(II)-replete conditions are required for CP to inhibit the growth of both Gram-negative and -positive bacteria. Taken together, these data provide a working model whereby calprotectin responds to physiological Ca(II) gradients to become a potent Zn(II) chelator in the extracellular space.

Figure 1

Proposed antimicrobial mechanism and structural features of human CP. (A) Proposed mechanism of action. CP is released from the neutrophil into the extracellular milieu where it competes with bacterial metal-ion transporters for bioavailable zinc and manganese. (B) and (C) The two putative transition metal-binding sites of human CP revealed at the dimer interface by x-ray crystallography (PDB: 1XK4). S100A8 is colored green and S100A9 is colored blue, and the putative metal-binding residues are shown in orange. Panel B illustrates the His₃Asp site (site 1) and panel C depicts the His₄ site (site 2). See ref. and Figure S1 of Supporting Information for the complete structure. (D) Amino acid alignment of the human calprotectin subunits S100A8 and S100A9 with human S100A7 and human S100A12. Alpha helices I–IV, the calcium-binding loops, and linker regions of S100A8 and S100A9 are color-coded and depicted above the alignments. The disordered C-terminus of S100A9 is underlined. The identified (A7, A12) and putative (A8, A9) metal-binding residues are highlighted in orange. The cysteine residues of S100A8 and S100A9 that were mutated to serine in this work are colored in red. Both S100A7 and S100A12 form homodimers, coordinate transition metals by interfacial His₃Asp motifs, and are involved in the innate immune response.

Figure 2

Cobalt binding to CP monitored by optical absorption spectroscopy. CP (400 μM) was titrated with 0 – 5 equiv of Co(II) at pH 7.0 (75 mM HEPES, 100 mM NaCl) and 25 °C. (A) Titration of CP-Ser with Co(II). A d—d transition centered at 556 nm (ε = 480 M⁻¹cm⁻¹) is observed. (B) Titration of CP-Ser ΔHis₄ with Co(II). A d—d transition centered at 556 nm (ε = 280 M⁻¹cm⁻¹) is observed. (C) Titration of CP-Ser ΔHis₃Asp with Co(II). A d—d transition centered at 499 nm (ε = 38 M⁻¹cm⁻¹) is observed and an expanded version of the plot is given in Figure S29. These titrations indicate that Co(II) binds to both the His₃Asp and His₄ sites of CP.

Figure 3

Low-temperature EPR spectroscopic signals of Co(II)-bound CP. (A) EPR spectra of 1.1 mM CP-Ser and the indicated metal-binding site mutants in the presence of 0.8 equiv of Co(II) at pH 7.0 (20 mM HEPES, 100 mM NaCl). The colors of the CP-Ser and ΔHis₄ spectra indicate the color of the samples following Co(II) addition. The samples of ΔHis₃Asp, and ΔΔ, in addition to the Co(II) in buffer reference, were colorless. Instrument conditions: temperature, 10.6 K; microwaves, 2 mW at 9.38 GHz; modulation amplitude, 8.0 G. (B) The black trace is a linear combination of the ΔHis₄ and ΔHis₃Asp spectra exhibited in A. Combination of ½ ΔHis₄ and ½ ΔHis₃Asp closely reproduces the spectrum of CP-Ser shown in purple. (C) Power saturation experiments. EPR spectra of the CP-Ser sample from A were recorded at 10.6 K with powers of 0.6, 2, 6, and 20 mW. The grey scale indicates increasing power from black to light grey.

Figure 4

Optical absorption spectra revealing displacement of Co(II) from 400 μM CP-Ser by Zn(II) addition at pH 7.0 (75 mM HEPES, 100 mM NaCl) and T = 25 °C. Red line: Optical absorption spectrum of CP-Ser in the presence of one equivalent of Co(II). Black lines: Optical absorption spectrum immediately after the addition of one equivalent of Zn(II) and at t = 15, 30, 60, 90 min after addition of Zn(II). Blue spectra: Optical absorption spectra immediately (top) and 60 min (bottom) after addition of a second equivalent of Zn(II).

Figure 5

Zinc response of 25 μM Zincon to in the presence of ca. 10 μM CP at pH 7.5 (75 mM HEPES, 100 mM NaCl) and 25 °C. The absorption values at 621 nm indicate that Zincon only responds to Zn(II) after CP-Ser binds two equivalents (red circles) or after ΔHis₄ and ΔHis₃Asp each coordinate one equivalent of Zn(II) (green triangles and blue squares, respectively). Negligible attenuation of the Zincon response is observed in the presence of ΔΔ (black diamonds).

Figure 6

Fluorescence response of 2 μM FZ3 to 2 μM Zn(II) in the presence of 10 μM CP at pH 7.5 (75 mM HEPES, 100 mM NaCl) and 25 °C. (A) No fluorescence change is observed following addition of Zn(II) to mixtures of FZ3 and CP-Ser. Inset: Expansion of the y-axis. Dotted black line, FZ3 emission in the absence of Zn(II); solid red line, FZ3 emission after addition of Zn(II). (B) Fluorescence enhancement is observed for FZ3 in the presence of ΔHis₄ (~2-fold, green line) and ΔHis₃Asp (~17-fold, blue line). FZ3 in the absence of any protein (e.g. no CP, black line) exhibits ~40-fold fluorescence turn-on following addition of one equivalent of Zn(II). The maximum emission for FZ3 in the presence of one equiv of Zn(II) was adjusted to an integrated emission value of 100 and the remaining emission spectra scaled accordingly.

Figure 7

Fluorescence response of 2 μM ZP4 to Zn(II) in the presence of 10 μM CP-Ser at pH 7.5 (75 mM HEPES, 100 mM NaCl) and 25 °C. Each emission spectrum was integrated and the resulting values were normalized to the maximum response and plotted against equivalents of Zn(II) per CP-Ser (αβ). The black squares indicate the titration performed in the absence of Ca(II). The red circles indicate the titration performed in the presence of 200 μM Ca(II). A representative titration for each set of conditions is shown. The titrations were fit to a two binding-site model. The black and red lines represent the fits, which afforded K_d1 = 133 ± 58 (−Ca), =10 pM (+ Ca) and K_d2 = 185 ± 219 nM (−Ca), ≥240 pM (+Ca). The K_d1 and K_d2 values are assigned to the His₃Asp and His₄ sites, respectively. Excitation was provided at 495 nm and the emission spectra were integrated from 505 – 650 nm. The corresponding titrations for ΔHis₃Asp and ΔHis₄ are presented in Figure S35.

Figure 8

Fluorescence response of 2 μM ZP4 to Zn(II) in the presence of ~5 μM CP-Ser (black circles), the (A8)H27A/(A9)H91A double mutant (green square), or the (A8)D30A single mutant (blue triangles). Each emission spectrum was integrated and the resulting values were normalized to the maximum response. For all titrations, excitation was provided at 495 nm and the emission spectra were integrated from 505 – 650 nm.

Figure 9

Competition experiments with ZP4 and CP-Ser at pH 7.5 (75 mM HEPES, 100 mM NaCl) and 25 °C. (A) Fluorescence response of 2 μM ZP4 following addition of 200 μM Ca(II) to mixtures of Zn(II):ZP4 and CP. Solutions of ZP4 and CP were titrated with Zn(II) until maximum turn-on of ZP4 was observed (data not shown) and Ca(II) was subsequently added (t = 0 min). Black circles: 10.6 μM ΔΔ; green circles: 9.4 μM ΔHis₄; red circles: 5.8 μM CP-Ser; blue circles: 7.4 μM ΔHis₃Asp. The normalized integrated emission value of 1 corresponds to maximum emission from ZP4. (B) Integrated emission from 1.9 μM ZP4 in the presence of 1:2 Zn(II):CP-Ser. Black circles, ZP4 only; blue circles, ZP4 and CP-Ser; red circles, ZP4 and 1:2 Zn(II):CP-Ser. Addition of 50 μM Zn(II) to the solutions at t = 300 min resulted in ZP4 turn-on as indicated by the data points at y ~ 3 × 10⁷. Excitation was provided at 495 nm and the emission spectra were integrated from 500 – 650 nm.

Figure 10

Wild-type CP and CP-Ser exhibit calcium-dependent antibacterial action for both Gram-negative and Gram-positive species. (A) Enterobacter aerogenes; (B) Escherichia coli; (C) Staphylococcus aureus. The black traces are cultures treated with CP (circles) or CP-Ser (squares) in the absence of a 2 mM Ca(II) supplement. The red traces are for cultures treated with CP (circles) or CP-Ser (squares) in the presence of a 2 mM Ca(II) supplement. The OD₆₀₀ values were recorded at t = 24 h (mean ± SEM for three independent replicates).

Figure 11

Growth inhibitory activity of 500 μg/mL wild-type CP, CP-Ser and the metal-binding site mutants. (A) Enterobacter aerogenes; (B) Escherichia coli; (C) Staphylococcus aureus. The cultures were incubated with 500 μg/mL CP in the presence of a 2 mM Ca(II) supplement (t = 30 °C). The OD₆₀₀ values (mean ± SEM for three independent replicates) were recorded at t = 8 (grey bars) and 24 h (white bars).