Design and characterization of calprotectin tetramerization variants for probing the role of oligomerization in receptor activation

Calprotectin is a heterodimer of the S100A8 and S100A9 EF-hand calcium binding proteins, which activates cell surface receptors that signal through the NF-κB inflammatory signaling pathway. Like all S100 proteins, calcium-induced conformational changes in calprotectin are required for binding to partner proteins. In the case of calprotectin, the addition of calcium correlates with the formation of a dimer of heterodimers (heterotetramer). Ligand-induced receptor oligomerization has been proposed as a mechanism of receptor activation. Conversely, it has also been suggested that calprotectin tetramerization can inhibit binding to receptors and serve as an autoinhibitory mechanism. In order to investigate the biological relevance of calprotectin tetramerization and facilitate in-depth biophysical and structural analysis, we have prepared three tetramerization-deficient variants: two single-site S100A8 mutations of hydrophobic isoleucine residues mediating the tetramer interface to lysine (I60K, I73K) and the corresponding double-site mutant (I60K/I70K). Dynamic light scattering, small-angle x-ray scattering, and nuclear magnetic resonance spectroscopy showed that all three tetramer-deficient variants remain as dimers in solution even in the presence of 40-fold excess calcium and undergo calcium-induced conformational changes. The crystal structure of I73K was determined to atomic-level resolution and confirms that the mutations cause only subtle, localized effects on the structure. Together, the results indicate that these tetramerization-deficient mutants will be useful reagents for discerning the functional role of calprotectin oligomerization in the activation of inflammatory receptors.