Functional modulation of RAGE activation by multimeric S100B using single-domain antibodies.

S100B is a multifunctional protein primarily found in the brain, where it plays crucial roles in cell proliferation, differentiation, and survival. It has intracellular and extracellular functions and, depending on S100B levels, can exhibit both neurotrophic and neurotoxic activities, both mediated by the receptor for advanced glycation end products (RAGEs). Here, we report the discovery and characterization of nanobodies (Nbs) targeting dimeric and tetrameric S100B, which are the two most abundant oligomeric functional forms of the protein, aiming to modulate S100B-mediated RAGE activation. Two Nbs were selected for detailed structural and functional studies and found to bind tetrameric S100B with high affinity, as determined by biolayer interferometry (BLI) analysis and size-exclusion chromatography-stable binary complex formation. Structural and docking analyses revealed preferential contact sites of Nbs with S100B regions implicated in interactions with RAGE, namely residues at the interfacial cleft on dimeric S100B and at hydrophobic cleft formed by the association of two homodimeric units in the tetramer. In accordance, assays in SH-SY5Y cells showed that Nbs modulate the RAGE-mediated neurotrophic activity of S100B by hindering its functional interactions with the receptor. BLI competition assays between tetrameric S100B and the RAGE-VC1 domain confirmed that Nbs selectively block S100B-mediated RAGE engagement, in agreement with cell activation experiments. These findings highlight Nbs as powerful tools for elucidating molecular and cellular mechanisms through the modulation of S100B and RAGE functions, inspiring potential therapeutic applications.