Biophysical Examination of the Calcium-Modulated Nickel-Binding Properties of Human Calprotectin Reveals Conformational Change in the EF-Hand Domains and His₃Asp Site

Calprotectin (CP, S100A8/S100A9 oligomer, MRP-8/MRP-14 oligomer) is a host-defense protein that sequesters nutrient transition metals from microbes. Each S100A8/S100A9 heterodimer contains four EF-hand domains and two transition-metal-binding sites. We investigate the effect of Ca­(II) ions on the structure and Ni­(II)-binding properties of human CP. By employing energy dispersive X-ray (EDX) spectroscopy, we evaluate the metal content of Ni­(II)-bound CP-Ser [oligomer of S100A8­(C42S) and S100A9­(C3S)] crystals obtained in the absence and presence of Ca­(II). We present a 2.1 Å resolution crystal structure of Ni­(II)-bound CP-Ser and compare this structure to a reported Ni­(II)- and Ca­(II)-bound CP-Ser structure [Nakashige, T. G., et al. (2017) <i>J. Am. Chem. Soc.</i> <i>139</i>, 8828–8836]. This analysis reveals conformational changes associated with coordination of Ca­(II) to the EF-hands of S100A9 and that Ca­(II) binding affects the coordination number and geometry of the Ni­(II) ion bound to the His₃Asp site. In contrast, negligible differences are observed for the Ni­(II)-His₆ site in the absence and presence of Ca­(II). Biochemical studies show that, whereas the His₆ site has a thermodynamic preference for Ni­(II) over Zn­(II), the His₃Asp site selects for Zn­(II) over Ni­(II), and relatively rapid metal exchange occurs at this site. These observations inform the working model for how CP withholds nutrient metals in the extracellular space