Investigation of Cu(II) Binding by Human Calprotectin Reveals an Unusual Fluxional Coordination Environment

Abstract Human calprotectin (CP, S100A8/S100A9 heterooligomer) is a multi-metal sequestering protein that contributes to the innate immune response by starving invading microbial pathogens of essential metal nutrients. Despite the ability of CP to withhold copper from microbial pathogens, its copper coordination chemistry is largely unexplored. Here we elucidate the Cu(II)-binding properties of CP-Ser [S100A8(C42S)/S100A9(C3S) variant] under conditions of high and low Ca(II) and present a working model for how CP sequesters Cu(II). Our biochemical and spectroscopic studies reveal that both the His3Asp and His6 sites bind Cu(II) with Ca(II)-dependent affinity, and Cu(II) binding to both sites is required for Cu(II)-dependent self-association of CP heterodimers to heterotetramers. Moreover, our spectroscopic data demonstrate that Cu(II) binds to the His6 site in a fluxional coordination environment, and that the mode of Cu(II) binding is allosterically modulated by Ca(II) coordination to the EF-hand domains and Cu(II) binding at the His3Asp site. Sequestration of Cu(II) using a fluxional His6 site contrasts with how CP captures other nutrient M(II) (M = Mn, Fe, Ni, Zn) at this site using a static and nearly idealized octahedral binding mode. This study reveals the remarkable ability of a fluxional site to participate in metal sequestration at the host–pathogen interface.