Characterization of the Calcium-Mediated Tetramerization of Human Calprotectin

Abstract

Calprotectin (CP) is an S100 protein that plays a role in the inflammatory response by acting as a ligand for the receptor for advanced glycation end-products (RAGE) and Toll-like receptor 4 (TLR-4). Activation of these receptors leads to upregulation of inflammatory cytokines, chemokines, and CP through the NF-κB pathway. In the case of people with irritable bowel disease (IBD) these pathways are stimulated which leads to damage of the gastrointestinal tract and disease symptoms. CP is a heterodimer of the S100A8 and S100A9 EF-hand calcium binding proteins. Like all S100 proteins, calcium-induced conformational changes in CP are required for binding to partner proteins. However, in the case of CP, the addition of calcium correlates with self-association and 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 CP tetramerization can inhibit binding to receptors and serve as an autoinhibitory mechanism to modulate the inflammatory response. In order to investigate the functional relevance of CP tetramerization and facilitate in-depth biophysical and structural analysis, I prepared single-site mutations of two hydrophobic residues mediating the tetramer interface (Ile60, Ile73) and a double-site mutant (I160K,170K). With the goal of maximally destabilizing the tetramer interface, these Ile residues were mutated to Lys (I60K, I73K). This work reports biophysical characterization of the three tetramer-deficient mutants using calorimetric, scattering, and structural approaches. Dynamic light scattering, small-angle x-ray scattering, and nuclear magnetic resonance show that the CP tetramer-deficient mutants remain as dimers in solution even in the presence of 40-fold excess calcium. The crystal structure of CP I73K mutant was determined to atomic level resolution and confirms there are virtually no differences in the structure of the heterodimer. Together, these results indicate that the mutants will be useful reagents for discerning the functional role of CP oligomerization in activation of cell-surface receptors. The implications of these results are discussed in the context of the complexity of CP receptor signaling.