肿瘤坏死因子受体超家族(TNFRSF)的成员是重要的治疗靶点，可以被激活以诱导癌细胞死亡或刺激免疫细胞增殖。虽然早已涉及这些受体组装先前配体关联状态，在人类疾病中需要优势干扰，但该状态迄今尚未进行结构表征。在这里，我们发现TNFRSF的代表成员死亡受体5(DR5-ECD)的外泌体，可以特异性地自我关联，当锚定到脂双层时，我们报道了核磁共振(NMR)确定的这种自我关联结构。意外的是，我们鉴定了两个非重叠的相互作用界面，可以传播到更高阶簇。基于结构的突变说明观察到的先前配体结构在表达DR5的细胞上被代表。DR5的先前配体关联担任自我抑制作用，因为部分解离前配体簇的单域抗体(sdAbs)可以使受体对其配体TRAIL变得更敏感，甚至在缺少TRAIL的情况下引起显著的受体信号。与需要多价结合聚集受体以激活的大多数促进性抗体不同，这些促进性sdAbs是单价的，专门针对受体的寡聚体自我抑制构型。我们的数据表明，像DR5这样的受体可以形成结构定义的先前簇，与信号不相容，并且真正的激动剂应该破坏先前配体簇，同时将其转化为信号产物簇。这种机制增强了我们对TNFRSF信号传导中长期存在的问题的理解，并暗示了通过针对受体前配体聚类来开发促进性分子的新机会。©2022年。作者(与中心卓越分子细胞科学)独家授权。

Members of the tumor necrosis factor receptor superfamily (TNFRSF) are important therapeutic targets that can be activated to induce death of cancer cells or stimulate proliferation of immune cells. Although it has long been implicated that these receptors assemble preligand associated states that are required for dominant interference in human disease, such states have so far eluded structural characterization. Here, we find that the ectodomain of death receptor 5 (DR5-ECD), a representative member of TNFRSF, can specifically self-associate when anchored to lipid bilayer, and we report this self-association structure determined by nuclear magnetic resonance (NMR). Unexpectedly, two non-overlapping interaction interfaces are identified that could propagate to higher-order clusters. Structure-guided mutagenesis indicates that the observed preligand association structure is represented on DR5-expressing cells. The DR5 preligand association serves an autoinhibitory role as single-domain antibodies (sdAbs) that partially dissociate the preligand cluster can sensitize the receptor to its ligand TRAIL and even induce substantial receptor signaling in the absence of TRAIL. Unlike most agonistic antibodies that require multivalent binding to aggregate receptors for activation, these agonistic sdAbs are monovalent and act specifically on an oligomeric, autoinhibitory configuration of the receptor. Our data indicate that receptors such as DR5 can form structurally defined preclusters incompatible with signaling and that true agonists should disrupt the preligand cluster while converting it to signaling-productive cluster. This mechanism enhances our understanding of a long-standing question in TNFRSF signaling and suggests a new opportunity for developing agonistic molecules by targeting receptor preligand clustering.© 2022. The Author(s) under exclusive licence to Center for Excellence in Molecular Cell Science, CAS.