Calcium Ions Tune the Zinc-Sequestering Properties and Antimicrobial Activity of Human S100A12

Abstract

Human S100A12 is a host-defense protein expressed and released by neutrophils that contributes to innate immunity. Apo S100A12 is a 21-kDa antiparallel homodimer that harbors two Ca(II)-binding EF-hand domains per subunit and exhibits two His₃Asp motifs for chelating transition metal ions at the homodimer interface. In this work, we present results from metal-binding studies and microbiology assays designed to ascertain whether Ca(II) ions modulate the Zn(II)-binding properties of S100A12 and further evaluate the antimicrobial properties of this protein. Our metal depletion studies reveal that Ca(II) ions enhance the ability of S100A12 to sequester Zn(II) from microbial growth media. We report that human S100A12 has antifungal activity against Candida albicans, C. krusei, C. glabrata and C. tropicalis, all of which cause human disease. This antifungal activity is Ca(II)-dependent and requires the His₃Asp metal-binding sites. We expand upon prior studies of the antibacterial activity of S100A12 and report Ca(II)-dependent and strain-selective behavior. S100A12 exhibited in vitro growth inhibitory activity against Listeria monocytogenes. In contrast, S100A12 had negligible effect on the growth of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1. Loss of functional ZnuABC, a high-affinity Zn(II) import system, increased the susceptibility of E. coli and P. aeruginosa to S100A12, indicating that S100A12 deprives these mutant strains of Zn(II). To evaluate the Zn(II)-binding sites of S100A12 in solution, we present studies using Co(II) as a spectroscopic probe and chromophoric small-molecule chelators in Zn(II) competition titrations. We confirm that S100A12 binds Zn(II) with a 2:1 stoichiometry, and our data indicate sub-nanomolar affinity binding. Taken together, these data support a model whereby S100A12 uses Ca(II) ions to tune its Zn(II)-chelating properties and antimicrobial activity.

Fig. 1. Crystal structures of metal-bound human S100A12 and amino acid sequence alignment of select S100A12 orthologues. (A) Structure of the Ca(ii)- and Cu(ii)-bound S100A12 homodimer (PDB ; 1ODB). Cu(ii) ions are shown as teal spheres, and Ca(ii) ions as yellow spheres. (B) View of the His₃Asp motif of S100A12 taken from the structure of a Zn(ii)-bound form (PDB ; 2WCB). The Zn(ii) ion is shown as a brown sphere. (C) Structure of the Zn(ii)-bound S100A12 homodimer (PDB ; 2WCB). Zn(ii) ions are shown as brown spheres. (D) Sequence alignment of human (h), porcine (p), and bovine (b) S100A12. The secondary structural elements presented above the alignment are for the human form. The transition-metal binding residues are presented in orange.

Fig. 2. S100A12 depletes Zn(ii) from microbial growth media. (A) Metal analysis of YPD/Tris medium treated with 0–250 μg mL^–1 S100A12 or 250 μg mL^–1 S100A12 ΔHis₃Asp. (B) Metal analysis of TSB/Tris medium treated with 0–250 μg mL^–1 S100A12 or 250 μg mL^–1 S100A12 ΔHis₃Asp. The experiments were conducted in the absence (light gray bars) and presence (dark gray bars) of a ≈2 mM Ca(ii) supplement (mean ± SEM, n ≥ 3).

Fig. 3. Human S100A12 exhibits Ca(ii)-dependent antifungal activity in YPD/Tris medium. (A) C. albicans SC 5314, (B) C. glabrata ATCC 200918, (C) C. krusei ATCC 200917, and (D) C. tropicalis ATCC MYA-3404. Black, Candida spp. treated with S100A12 in the absence of a Ca(ii) supplement. Red, Candida spp. treated with S100A12 in the presence of a ≈2 mM Ca(ii) supplement. The OD₆₀₀ values were recorded at t = 30 h (mean ± SEM, n = 3).

Fig. 4. The His₃Asp motifs of S100A12 are essential for antifungal activity against C. albicans. (A) Antifungal activity of 1000 μg mL^–1 S100A12 and S10012 ΔHis₃Asp (t = 20 h, mean ± SEM, n = 3). (B) Antifungal activity of 125 μg mL^–1 S100A12 and Zn(ii)–S100A12 (t = 30 h, mean ± SEM, n = 3). The Zn(ii)–S100A12 sample was prepared by pre-incubating S100A12 with 2 equiv. of Zn(ii) (11.9 μM) prior to the assay. The bar labeled “Zn(ii)” indicates the growth of a culture where 11.9 μM Zn(ii) was added to the YPD/Tris medium. For each assay, the YPD/Tris medium contained a ≈2 mM Ca(ii) supplement.

Fig. 5. Antibacterial activity of human S100A12 (1000 μg mL^–1) against S. aureus ATCC 25923 (Sa), E. coli K-12 (Ec), P. aeruginosa PAO1 (Pa), L. monocytogenes ATCC 19115 (Lm), and L. plantarum WSCF1 (Lp) in the absence and presence of a ≈2 mM Ca(ii) supplement. The assays were performed in TSB/Tris medium (t = 20 h, T = 30 °C).

Fig. 6. S100A12 exhibits antibacterial activity against E. coli with defective Zn(ii) uptake machinery in TSB/Tris medium. (A) Antibacterial activity of S100A12 against E. coli K-12. (B) Antibacterial activity of S100A12 against E. coli K-12 ΔznuA. Black, E. coli treated with S100A12 in the absence of a Ca(ii) supplement. Red, E. coli treated with S100A12 in the presence of ≈2 mM Ca(ii). The OD₆₀₀ values were recorded at t = 20 h (mean ± SEM, n = 3).

Fig. 7. S100A12 metal-binding titrations. (A) Optical absorption spectra of S100A12 (200 μM) titrated with 0–5 equiv. of Co(ii) at pH 7.0 (75 mM HEPES, 100 mM NaCl) and 25 °C. (B) Plot of absorbance at 550 nm versus equivalents of Co(ii) added for titration of S100A12 or ΔHis₃Asp (200 μM). (C) Response of Zincon (25 μM) to Zn(ii) in the presence of S100A12 or ΔHis₃Asp (10 μM) at pH 7.0 (75 mM HEPES, 100 mM NaCl) and 25 °C (mean ± SEM, n = 3). The Zn(ii)–Zincon complex absorbs at 621 nm. (D) Response of MF2 (10 μM) to Zn(ii) in the presence of S100A12 or ΔHis₃Asp (10 μM) at pH 7.0 (75 mM HEPES, 100 mM NaCl) and 25 °C (mean ± SEM, n = 3). The Zn(ii)–MF2 complex absorbs at 325 nm. Plots for Co(ii) titrations and Zincon competitions performed in the presence of Ca(ii) are presented in Fig. S7 and S9.