S100B is a multifunctional protein primarily found in the brain, where it plays crucial roles in cell proliferation, differentiation, and survival. It has intra- 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 (RAGE). 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 analysis and SEC-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 the 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. Biolayer interferometry 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.

中文翻译：

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使用单域抗体通过多聚体 S100B 对 RAGE 激活进行功能调节。


S100B 是一种主要存在于大脑中的多功能蛋白，在细胞增殖、分化和存活中起着至关重要的作用。它具有细胞内和细胞外功能，并且根据 S100B 水平，可以表现出神经营养和神经毒性活性，这两者都是由晚期糖基化终末产物受体 （RAGE） 介导的。在这里，我们报告了靶向二聚体和四聚体 S100B 的纳米抗体 （Nbs） 的发现和表征，这是蛋白质的两种最丰富的寡聚功能形式，旨在调节 S100B 介导的 RAGE 激活。选择两个 Nbs 进行详细的结构和功能研究，并通过生物膜干涉分析和 SEC 稳定的二元复合物形成确定，发现它们以高亲和力结合四聚体 S100B。结构和对接分析揭示了 Nbs 与 S100B 区域的优先接触位点，这些区域与 RAGE 相互作用有关，即二聚体 S100B 界面裂隙处的残基和由四聚体中两个同型二聚体单元结合形成的疏水裂隙处的残基。相应地，SH-SY5Y 细胞中的检测表明，Nbs 通过阻碍 S100B 与受体的功能相互作用来调节 RAGE 介导的神经营养活性。四聚体 S100B 和 RAGE-VC1 结构域之间的生物膜干涉竞争测定证实，Nbs 选择性阻断 S100B 介导的 RAGE 参与，与细胞活化实验一致。这些发现突出了 Nbs 是通过调节 S100B 和 RAGE 功能来阐明分子和细胞机制的强大工具，从而激发了潜在的治疗应用。