Antibodies against N-methyl-d-aspartate receptors (NMDARs) are most frequently detected in persons with autoimmune encephalitis (AE) and used as diagnostic biomarkers. Elucidating the structural basis of monoclonal antibody (mAb) binding to NMDARs would facilitate the development of targeted therapy for AE. Here, we reconstructed nanodiscs containing green fluorescent protein-fused NMDARs to label and sort individual immune B cells from persons with AE and further cloned and identified mAbs against NMDARs. This allowed cryo-electron microscopy analysis of NMDAR–Fab complexes, revealing that autoantibodies bind to the R1 lobe of the N-terminal domain of the GluN1 subunit. Small-angle X-ray scattering studies demonstrated NMDAR–mAb stoichiometry of 2:1 or 1:2, structurally suitable for mAb-induced clustering and endocytosis of NMDARs. Importantly, these mAbs reduced the surface NMDARs and NMDAR-mediated currents, without tonically affecting NMDAR channel gating. These structural and functional findings imply that the design of neutralizing antibody binding to the R1 lobe of NMDARs represents a potential therapy for AE treatment.

抗 N-甲基-d-天冬氨酸受体 （NMDAR） 的抗体最常在自身免疫性脑炎 （AE） 患者中检测到，并用作诊断生物标志物。阐明单克隆抗体 （mAb） 与 NMDAR 结合的结构基础将有助于开发 AE 靶向治疗。在这里，我们重建了含有绿色荧光蛋白融合 NMDAR 的纳米圆盘，以标记和分选来自 AE 患者的单个免疫 B 细胞，并进一步克隆和鉴定了针对 NMDAR 的 mAb。这允许对 NMDAR-Fab 复合物进行冷冻电子显微镜分析，揭示自身抗体与 GluN1 亚基 N 末端结构域的 R1 叶结合。小角 X 射线散射研究表明，NMDAR-mAb 化学计量为 2：1 或 1：2，结构适用于 mAb 诱导的 NMDAR 聚集和内吞作用。重要的是，这些 mAb 减少了表面 NMDAR 和 NMDAR 介导的电流，而不会影响 NMDAR 通道门控。这些结构和功能发现表明，与 NMDAR 的 R1 叶结合的中和抗体的设计代表了 AE 治疗的潜在疗法。