Calcium ions tune the zinc-sequestering properties and antimicrobial activity of human S100A12

Human S100A12 exhibits Ca(ii)-dependent Zn(ii)-binding properties and antifungal activity.


Experimental Section
Materials and General Methods. All solvents and chemicals were obtained from commercial suppliers and used as received. All aqueous solutions were prepared using Milli-Q water (18.2 MΩcm, 0.22-µm filter).
For the microbiology assays, Luria-Bertani (LB) medium, Tryptic Soy Broth (TSB) medium, and Yeast Peptone Dextrose (YPD) medium, deMan, Rogosa and Sharpe (MRS) medium, and Brain Heart Infusion (BHI) medium, and agar plates were prepared with Milli-Q water. A Beckman Coulter DU 800 spectrophotometer thermostatted at 25 o C with a Peltier temperature controller or an Agilent 8453 diode-array spectrophotometer controlled with manufacturer-supplied software and thermostatted at 25 o C by a circulating water bath were employed for the OD 600 measurements of bacterial and fungal cultures.
For metal-binding experiments, HEPES buffer was prepared with Ultrol grade HEPES (free acid, Calbiochem) and TraceSELECT NaCl (Sigma), and TraceSELECT aqueous NaOH (Sigma) was used to adjust the pH. To reduce metal-ion contamination, Teflon-coated or plastic spatulas were used to transfer buffer reagents, and buffers were treated with Chelex 100 resin (Bio-Rad, 10 g/L) by stirring in a polypropylene beaker for at least 1 h. The Chelex resin was removed by passing the buffer through a 0.22-µm filter, and all buffers were stored in polypropylene containers. All metal-binding studies were conducted at pH 7.0 in 75 mM HEPES, 100 mM NaCl. A Tris buffer (1 mM Tris, pH 7.5) prepared from Tris base (J. T. Baker) was used for circular dichroism (CD) spectroscopy experiments. This buffer was treated with Chelex resin for 1 h (10 g/L), filtered through a 0.22-µm filter, and the pH was readjusted to 7.0 with hydrochloric acid.
Cobalt stock solutions (100 mM) were prepared from 99.999% CoCl 2 hydrate (Sigma) and Milli-Q water, and zinc stock solutions (100 mM) were prepared from 99.999% anhydrous ZnCl 2 (Sigma) and Milli-Q water. The metal stock solutions were prepared in acid-washed volumetric glassware and transferred to sterile polypropylene tubes for long-term storage. The working solutions were prepared by diluting the stock solutions in Milli-Q water.
Stocks of FZ3 (≈2 mM), and MF2 (≈2 mM), were prepared in Chelexed-treated Milli-Q water, aliquoted into 50-µL portions, and stored at -20 o C. Each aliquot was thawed only once, and experiments with these reagents were performed in the dark.
The pET41a-S100A12 expression plasmid was previously described. 1  The reconstitution and purification of human S100A12 and S100A12 ΔHis 3 Asp were performed following a modified published protocol. 1 For larger-scale protein preparations, the purification protocol was modified to avoid pressure limit issues with the ÄKTA Purifier FPLC system (GE Life Sciences). The complete modified protocol is detailed below.
Frozen cell pellets (≈3 g from a 2-L culture) were thawed on ice for ≈20 min and resuspended in 10 mL/g of lysis buffer (50 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0.5% Triton X-100). The resulting suspension was sonicated on ice using a Branson sonicator (40% amplitude for 2.5 min, 30 sec on / 10 sec off), and the crude lysate for separation of homodimeric S100A12 from higher oligomeric and aggregated species of S100A12. Fractions containing S100A12 (as determined by SDS-PAGE, 15% Tris-HCl gel) were pooled, concentrated to ≈10 mL, and purified by size-exclusion chromatography (SEC) using a HiLoad 26/600 S75 pg column (GE Lifesciences). For purified protein that was used in metal-binding experiments, the running buffer of the SEC was 20 mM HEPES, 100 mM NaCl, pH 8.0. For protein that was employed in microbiology assays, the running buffer was 20 mM Tris, 100 mM NaCl, pH 7.5. Fractions containing S100A12 were pooled and dialyzed against 1 L of the appropriate buffer (20 mM HEPES, 100 mM NaCl, pH 8.0, or 20 mM Tris, 100mM NaCl, Protein Characterization. S100A12 and S100A12 ΔHis 3 Asp were characterized by SDS-PAGE, mass spectrometry, analytical size exclusion chromatography, and circular dichroism (CD) spectroscopy using reported procedures. 1
For these studies, two types of growth media were analyzed. The YPD/Tris medium was composed of a 32:68 (v:v) ratio of YPD and Tris buffer (20 mM Tris-HCl, 100 mM NaCl, pH 7.5).
The TSB/Tris medium was composed of a 32:68 (v:v) ratio of TSB and Tris buffer. Western Blot. To ensure that the S100A12 present during treatment of growth medium did not pass through the spin filter, Western blot analysis was performed on the retentate and flow through obtained from each sample. Sodium dodecyl sulfate-PAGE (SDS-PAGE) was performed on a 15% glycine gel. The proteins were transferred to a nitrocellulose membrane following the manufacturer's procedure (BioRad). S100A12 was blotted with a 1:1000 dilution of monoclonal mouse IgG2b to human S100A12/EN-RAGE (161205) (R&D Systems). The antibody was blotted with a 1:10,000 dilution of infrared dye-labeled goat anti-mouse IgG (LI-COR Biosciences), and the blot was visualized using a LI-COR Odyssey Scanner.
Antifungal Activity Assays. The growth inhibitory activity of S100A12  [S100A12] ratio. Zincon has reported K d,Zn values of 12.6 and 5.8 µM. 5,6 Zinc Competition with S100A12 and MF2. A 2-mL solution containing 10 µM S100A12 or S100A12 ΔHis 3 Asp and 10 µM MF2 was prepared in a quartz cuvette (75 mM HEPES, 100mM NaCl, pH 7.0) and titrated with 0-4 equivalents of Zn(II) (1 µL of a 2 mM ZnCl 2 aqueous solution per addition) at room temperature. The samples were allowed to equilibrate for 2 min after each Zn(II) addition, and the optical absorption spectra were collected from 200 to 800 nm.
The absorbance increase at 325 nm and decrease at 366 nm were plotted versus the [Zn(II)] / [S100A12] ratio. Reported K d,Zn values of MF-2 are 20 and 36 nM. 7,8 Zinc Competition with S100A12 and FZ3. Solutions (1 mL) containing FZ3 (2 µM) and S100A12 (2 µM) or S100A12 ΔHis 3 Asp (2 µM) were prepared in PMMA cuvettes (75 mM HEPES, 100 mM NaCl, pH 7.0). Each solution was mixed gently and incubated for 1 h in the dark at room temperature. The emission spectrum of each solution was then recorded. One equivalent of Zn(II) was subsequently added to each sample, and the solutions were gently mixed and incubated for 2.5 h in the dark at room temperature. The emission spectrum of each solution was then recorded. The samples were excited at 494 nm, and the emission was monitored from 500-650 nm (-Zn(II) samples) or 507-650 nm (+Zn(II) samples). Emission spectra from a representative trial are presented. The apparent K d,Zn of FZ3 is 9 nM. 9 Table S1. Molecular weights and extinction coefficients for S100A12 monomers.
The znuA gene is not annotated in the Seattle Pseudomonas aeruginosa PAO1 Mutant Library.
The znuA gene was found by searching "znuA" at pseudomonas.com. Gene with locus tag PA5498 is annotated with the name "znuA." BLAST analysis revealed that this gene is a homologue to other znuA genes. The Seattle Pseudomonas aeruginosa PAO1 Transposon Mutant Library was searched and five strains with a znuA mutation were found. One of these strains (mutant: phoAbp01q2A01, gene: PA5498) was employed in this study. The sequence of the ZnuA protein deduced from gene PA5498 for P. aeruginosa PAO1 was blasted against two proteins identified as ZnuA transporters (accession numbers EOT09947 and EOT14059) in P. aeruginosa PA14. ZnuA PAO1 was found to share 100% and 72% sequence homology with the ZnuA proteins EOT09947 and EOT14059, respectively. A recent report also identified PA5498 as ZnuA. 10 wild-type ΔznuA  Figure S10. S100A12 outcompetes FZ3 for Zn(II). Fluorescence response of 2 µM FZ3 to 2 µM Zn(II) in the presence of 2 µM S100A12 or S100A12 ΔHis 3 Asp at pH 7.0 (75 mM HEPES, 100 mM NaCl) and 25 °C. (A) An ≈36-fold fluorescent enhancement is observed for FZ3 in the absence of protein (blue trace) and in the presence of ΔHis 3 Asp (red trace) upon addition of 1 equiv. of Zn(II). Negligible fluorescent response is observed for FZ3 in the presence of S100A12 (green trace) upon addition of 1 equiv. of Zn(II) ( Inset: Expansion of the y-axis). (B) integrated emission values for the data presented in panel A and replicates. The maximum emission for FZ3 in the presence of 1 equiv. of Zn(II) was normalized to an integrated emission value of 100, and the remaining emission spectra were scaled accordingly (mean ± SEM, n=3).