S100 Proteins in the Innate Immune Response to Pathogens

Abstract

S100 proteins are distinct dimeric EF-hand Ca²⁺-binding proteins that can bind Zn²⁺, Mn²⁺, and other transition metals with high affinity at two sites in the dimer interface. Certain S100 proteins, including S100A7, S100A12, S100A8, and S100A9, play key roles in the innate immune response to pathogens. These proteins function via a "nutritional immunity" mechanism by depleting essential transition metals in the infection that are required for the invading organism to grow and thrive. They also act as damage-associated molecular pattern ligands, which activate pattern recognition receptors (e.g., Toll-like receptor 4, RAGE) that mediate inflammation. Here we present protocols for these S100 proteins for high-level production of recombinant protein, measurement of binding affinities using isothermal titration calorimetry, and an assay of antimicrobial activity.

Keywords: Antimicrobial growth assay; Calprotectin; Host-pathogen interaction; Inflammatory response; Isothermal titration calorimetry; Metal binding; Nutritional immunity; Protein expression; Protein purification; S100 proteins; S100A12; S100A7; S100A8; S100A9.

Fig. 1

The effect of Ca²⁺ binding on the structure of S100A12. The Ca²⁺-free protein (green and cyan) is shown on the left, and the Ca²⁺-bound state (magenta and orange) is shown on the right, superimposed on the Ca²⁺-free state. Note the change in position of helix 3 between the Ca²⁺-free and Ca²⁺-bound states. PDB ID: 2WCE and 2M9G

Fig. 2

Structure of Ca²⁺- and Mn²⁺-bound CP. S100A8 is colored red, and S100A9 is colored purple. Ca²⁺ ions are gray spheres and the Mn²⁺ ion is a blue sphere. The side chains chelating transition metal ions in S1 and S2 are shown. PDB ID: 4GGF

Fig. 3

The binding of Zn²⁺ to CP monitored by ITC. ITC isotherms of CP as Zn²⁺ are titrated from the syringe into the cell with S1 or S2 knockout mutants (ΔS1, ΔS2). Note the modest differences in K_d values between S1 and S2. These panels were taken from Fig. 1 in the original publication in Damo et al. [8]

Fig. 4

Representative growth assay of S. aureus in the presence of increasing concentrations of recombinant CP. Growth was quantified by 0.D.₆₀₀. Error bars represent the standard deviation of at least three replicates. Panel is adapted from the original publication in Corbin et al. [20]