Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) is a critical regulatory molecule in modulation of T cell immune responses. Here we report the mouse GITR (mGITR) and mGITR ligand (mGITRL) complex structure and find that the binding interface of mGITR and mGITRL is distinct from the typical tumor necrosis factor superfamily (TNFSF)/TNF receptor superfamily (TNFRSF) members. mGITR binds to its ligand with a single domain, whereas the binding interface on mGITRL is located on the side, which is distal from conserved binding sites of TNFSF molecules. Mutational analysis reveals that the binding interface of GITR/GITRL in humans is conserved with that in the mouse. Substitution of key interacting D93-I94-V95 (DIV) in mGITR with the corresponding K93-F94-S95 (KFS) in human GITR enables cross-recognition with human GITRL and cross-activation of receptor signaling. The findings of this study substantially expand our understanding of the interaction of TNFSF/TNFRSF superfamily molecules and can benefit the future design of biologics by targeting GITR/GITRL.

糖皮质激素诱导的肿瘤坏死因子受体家族相关蛋白 (GITR) 是调节 T 细胞免疫反应的关键调节分子。在这里，我们报告了小鼠 GITR (mGITR) 和 mGITR 配体 (mGITRL) 的复合结构，并发现 mGITR 和 mGITRL 的结合界面不同于典型的肿瘤坏死因子超家族 (TNFSF)/TNF 受体超家族 (TNFRSF) 成员。mGITR 通过单个结构域与其配体结合，而 mGITRL 上的结合界面位于侧面，远离 TNFSF 分子的保守结合位点。突变分析表明，人类中 GITR/GITRL 的结合界面与小鼠中的结合界面是保守的。将 mGITR 中的关键相互作用 D93-I94-V95 (DIV) 替换为人类 GITR 中相应的 K93-F94-S95 (KFS) 能够与人类 GITRL 交叉识别和受体信号传导的交叉激活。这项研究的结果极大地扩展了我们对 TNFSF/TNFRSF 超家族分子相互作用的理解，并且可以通过靶向 GITR/GITRL 来有益于生物制剂的未来设计。