Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry.

Although knowledge of the coordination chemistry and metal-withholding function of the innate immune protein human calprotectin (hCP) has broadened in recent years, understanding of its Ca-binding properties in solution remains incomplete. In particular, the molecular basis by which Ca binding affects structure and enhances the functional properties of this remarkable transition-metal-sequestering protein has remained enigmatic. To achieve a molecular picture of how Ca binding triggers hCP oligomerization, increases protease stability, and enhances antimicrobial activity, we implemented a new integrated mass spectrometry (MS)-based approach that can be readily generalized to study other protein-metal and protein-ligand interactions. Three MS-based methods (hydrogen/deuterium exchange MS kinetics; protein-ligand interactions in solution by MS, titration, and H/D exchange (PLIMSTEX); and native MS) provided a comprehensive analysis of Ca binding and oligomerization to hCP without modifying the protein in any way. Integration of these methods allowed us to (i) observe the four regions of hCP that serve as Ca-binding sites, (ii) determine the binding stoichiometry to be four Ca per CP heterodimer and eight Ca per CP heterotetramer, (iii) establish the protein-to-Ca molar ratio that causes the dimer-to-tetramer transition, and (iv) calculate the binding affinities associated with the four Ca-binding sites per heterodimer. These quantitative results support a model in which hCP exists in its heterodimeric form and is at most half-bound to Ca in the cytoplasm of resting cells. With release into the extracellular space, hCP encounters elevated Ca concentrations and binds more Ca ions, forming a heterotetramer that is poised to compete with microbial pathogens for essential metal nutrients.