Formation of the intradimer disulfide bond in human calprotectin maintains metal-withholding function and tunes proteolytic susceptibility.

Human calprotectin (CP, S100A8/S100A9 oligomer, MRP8/MRP14 oligomer) is a metal-sequestering protein that contributes to nutritional immunity. Each human CP subunit contains a single Cys residue-Cys42 in S100A8 and Cys3 in S100A9-and recent reports have revealed that these residues can undergo disulfide bond formation resulting in a covalently linked heterodimer species, hereafter referred to as disulfide-linked CP (dslCP). Nevertheless, the biochemical and functional consequences of this intradimer disulfide linkage are largely unknown. Here, we report a robust reconstitution and purification protocol affording dslCP and present initial biochemical and functional evaluation of the protein. Our investigations demonstrate that dslCP undergoes Ca(ii)-dependent self-association to form heterotetramers, depletes multiple metals from bacterial growth media, and induces an iron-starvation response in diverse bacterial pathogens. The intradimer disulfide linkage exhibits a midpoint potential of -213 mV, indicating that it can become oxidized in the extracellular space. Studies of enzymatic disulfide bond reduction reveal that divalent cation binding renders dslCP a poor substrate for the thioredoxin system. Investigations of proteolytic stability show that the intradimer disulfide linkage in dslCP enhances the susceptibility of the protein scaffold to digestion by human neutrophil elastase and trypsin, supporting a model wherein oxidative post-translational modifications direct protein lifetime. Our work expands upon the known roles of post-translational modifications of CP and highlights the need for further studies to define how oxidative modifications regulate the structure, stability, and function of this important host-defense protein.