Calcium Regulates S100A12 Zinc Sequestration by Limiting Structural Variations

Abstract

Antimicrobial proteins such as S100A12 and S100A8/A9 are highly expressed and secreted by neutrophils during infection and participate in human immune response by sequestering transition metals. At neutral pH, S100A12 sequesters Zn²⁺ with nanomolar affinity, which is further enhanced upon calcium binding. We investigated the pH dependence of human S100A12 zinc sequestration by using Co²⁺ as a surrogate. Apo-S100A12 exhibits strong Co²⁺ binding between pH 7.0 and 10.0 that progressively diminishes as the pH is decreased to 5.3. Ca²⁺ -S100A12 can retain nanomolar Co²⁺ binding up to pH 5.7. NMR spectroscopic measurements revealed that calcium binding does not alter the side-chain protonation of the Co²⁺ /Zn²⁺ binding histidine residues. Instead, the calcium-mediated modulation is achieved by restraining pH-dependent conformational changes to EF loop 1, which contains Co²⁺ /Zn²⁺ binding Asp25. This calcium-induced enhancement of Co²⁺ /Zn²⁺ binding might assist in the promotion of antimicrobial activities in humans by S100 proteins during neutrophil activation under subneutral pH conditions.

Keywords: NMR spectroscopy; S100 metalloproteins; S100A12; nutritional immunity; zinc sequestration.

Figure 1.

A) The primary sequence of human S100A12 showing Zn²⁺ binding residues (colored cyan) and EF hand loops. The secondary structure elements are presented above the sequence and displayed as blue bar for helices, pink arrows for β-strand and cyan line for loops; B) an expansion of Zn²⁺ bound S100A12 crystal structure showing the zinc binding site (PDB: 2WCB). The two monomers are colored blue and gray. The zinc and sodium ions are colored purple and blue, respectively and; C) an overlay of the crystal structures of apo S100A12 (PDB: 2WCF, blue) and Ca²⁺ bound S100A12 (PDB: 1E8A, orange). The EF-hand loops are colored using the same color scheme as in (A).

Figure 2.

Overlay of 14.1 T ¹H-¹⁵N HSQC spectra of the S100A12 at pH 8.3 (blue), 6.0 (pink) and 4.5 (light green) for A) apo-protein and, B) calcium bound S100A12. The residues involved in His₃Asp Zn²⁺ binding motif are labeled in red.

Figure 3.

pH dependence of Co²⁺ binding to S100A12 in the absence (orange) and presence (blue) of Ca²⁺ shown with a plot of absorbance at 556 nm versus pH. At pH values greater than 7.3 minor protein precipitation in the calcium bound S100A12 was observed that interfered with the baselines of the absorption spectra, giving rise to irregular absorption maxima under these conditions.

Figure 4.

¹H-¹⁵N HMQC spectra showing the tautomeric forms of histidine residues in (A) apo (16.8 T) and; (B) calcium bound S100A12 (14.1 T) at pH 7.5 (orange) and 6.0 (blue). The cross-peak patterns belonging to a single residue are marked with colored boxes.

Figure 5.

2D and 3D ¹H, ¹⁵N, ¹³C experiments conducted for the assignments of histidine sidechain nuclei of calcium bound S100A12 at pH 6.0. A) ¹H-¹⁵N HMQC; B) 2D ¹H-¹³C (HB)CB(CDCGCD)HD; C) a strip plot of the 3D HN(CO)CACB (blue) and HNCACB (red) spectra; D) a strip plot of the ¹H-¹⁵N NOESY-HSQC (orange) spectrum. Resonances associated with a residue are labeled and marked with same color across all spectra.

Figure 6.

Site specific absolute values of chemical shift perturbation between pH 6.0 and 7.0 for (A) C^α and, (B) N^H nuclei in the absence (pink) or presence (blue) of calcium ion. The negative values represent residues for which resonance assignments are not available. The secondary structure of the apo-protein determined from the crystal structure (PDB code 2WCF) is displayed at the top: α-helices (blue bars), β-strands (pink arrows), and loop regions (cyan lines). The values 1.38 and 3.33 in (B) refer to CSPs that are larger than the displayed range of the plot.

Figure 7.

SSP based C^α and N^H chemical shifts at pH 7.0 (red) and 6.0 (blue) for (A) Apo-S100A12 and, (B) Ca²⁺-S100A12. The secondary structure of the apoprotein determined from the crystal structure (PDB code 2WCF) is displayed at the top: α-helices (blue bars), β-strands (pink arrows), and loop regions (cyan lines).