The Molecular Basis for Calprotectin Function in Nutritional Immunity

Abstract

Background: The S100A8/S100A9 heterodimer calprotectin (CP) is expressed abundantly in neutrophils and plays a key role in innate immunity. In tissue abscesses, CP sequesters the transition metals manganese (Mn) and zinc (Zn) which serves to inhibit microbial growth and contributes to the control of pathogens. This activity is often referred to as nutritional immunity. We have shown previously that CP contains two transition metal binding sites, both of which have high affinity for Zn, however only one of which exhibits high affinity for Mn. Methods: Here we report an in-depth characterization of the biophysical and biochemical properties of CP-Mn and CP-Zn complexes using a combination of isothermal titration calorimetry (ITC), mutagenesis, and microbiology experiments. Atomic level details into the structural basis for function are provided by a high-resolution crystal structure of CP with bound Mn. Results: The crystal structure of CP with bound Mn has been determined to 1.8 A. ITC experiments show a stoichiometry of two Zn ions with dissociation constants of 1.4 nM and 5.6 nM, whereas only one Mn ion binds with high affinity (dissociation constant 1.3 nM). Mutants of CP with inactivated metal binding sites have been designed and characterized physically and functionally. Conclusions: CP binds two Zn ions, but only one Mn ion, with high affinity. The x-ray crystal structure reveals a single octahedrally coordinated Mn ion at the S100A8/S100A9 subunit interface that also involves the C-terminal tail of S100A9. The chelation of Mn by six histidine residues is unique and has not been observed in other Mn binding proteins. The biochemical and biophysical characterization of wild type and mutant CP variants that inhibit Mn binding show that sequestration of Mn is required for the antimicrobial activity of CP. The distinctive features of the Mn binding site explain why CP exhibits broad-spectrum anti-microbial activity via a nutritional immunity mechanism.