Cu(II) EPR Reveals Two Distinct Binding Sites and Oligomerization of Innate Immune Protein Calgranulin C

S100A12 or Calgranulin C is a homodimeric antimicrobial protein of the S100 family of EF-hand calcium-modulated proteins. S100A12 is involved in many diseases such as inflammation, tumor invasion, cancer and neurological disorders such as Alzheimer’s disease. The binding of transition metal ions to the protein is important as the sequestering of the metal ion induces conformational changes in the protein, inhibiting the growth of various pathogenic microorganisms. In this work, we probe the Cu²⁺ binding properties of Calgranulin C. We demonstrate that the two Cu²⁺ binding sites in Calgranulin C show different coordination environments in solution. Continuous wave-electron spin resonance (CW-ESR) spectra of Cu²⁺-bound protein clearly show two distinct components at higher Cu²⁺:protein ratios, which is indicative of the two different binding environments for the Cu²⁺ ions. The g_|| and A_|| values are also different for the two components, indicating that the number of directly coordinated nitrogen in each site differs. Furthermore, we perform CW-ESR titrations to obtain the binding affinity of the Ca²⁺-loaded protein to Cu²⁺ ions. We observe a positive cooperativity in binding of the two Cu²⁺ ions. To further probe the Cu²⁺ coordination, we also perform electron spin echo envelope modulation (ESEEM) experiment. We perform ESEEM at two different fields where one Cu²⁺ binding site dominates the other. At both sites we see distinct signatures of Cu²⁺–histidine coordination. However, we clearly see that the ESEEM spectra corresponding to the two Cu²⁺ binding sites are significantly different. There is clear change in the intensity of the double quantum peak with respect to the nuclear quadrupole interaction peak at the two different fields. Furthermore, ESEEM along with hyperfine sublevel correlation show that only one of the two Cu²⁺ binding sites has backbone coordination, confirming our previous observation. Finally, we perform double electron–electron resonance spectroscopy to probe if the difference in binding environment is due to the Cu²⁺ binding to different sites in the protein. We obtain a distance distribution with a sharp peak at ~ 3 nm and a broad peak at ~ 4 nm. The shorter distance agrees with the Cu²⁺–Cu²⁺ distance expected for a dimer from the crystal structure. The longer distance is consistent with the Cu²⁺–Cu²⁺ distance when oligomerization occurs.