Importance of cysteines for the binding of S100A6 to the RAGE receptor – Towards a first molecular model for S100 covalent dimerization

Extracellular S100 proteins act as alarmins and trigger pro-inflammatory signaling cascades by activating their cognate cell-surface receptor RAGE, thereby contributing to both normal and pathological inflammation depending on the physiological context. These ligand-receptor interactions occur in an oxidative environment that is known to induce post-translational modifications, notably on the cysteine residues present in S100 proteins. How cysteine oxidation affects the architecture of S100 proteins and their interaction with RAGE remains poorly understood as most in vitro studies employ cysteine mutants or reduced conditions. Using our model protein S100A6 and size exclusion chromatography-based binding assays in non-reducing conditions, we here demonstrate that the unique cysteine of S100A6, Cys3, is essential for the binding to RAGE. We further show that full complexation can be restored by introducing a cysteine at conserved position 84, where a Cys residue is found in at least ten other RAGE-binding S100 proteins. Structural analysis of the resulting complex between RAGE ectodomain and S100A6 mutant Y84C further reveals that the presence of Cys84 induces the formation of a covalent disulfide bond between the two S100A6 protomers, thus stabilizing the same RAGE-bound S100A6 conformation as with the WT protein. Finally, modeling of other S100 proteins in the RAGE-bound conformation suggests that this covalent S100 dimer architecture may be adopted by other members of the family, already reported to form disulfide-crosslinked oligomeric species. Altogether, our findings highlight the importance of S100 cysteines for the binding to RAGE and provide a first molecular model for S100 covalent homodimerization.