轴突初始片段(AIS)具有特征性的密集聚集的电压门控钠通道(Nav),细胞粘附分子Neurofascin186(Nfasc),神经元支架蛋白Ankyrin-G(AnkG),这有利于动作电位的产生和轴突极性的维持。然而,AIS装配的基础机制,维护,和可塑性仍然知之甚少。这里,我们报告了AnkG锚蛋白重复序列(ANK重复序列)结构域的高分辨率晶体结构,其结合位点在Nfasc细胞质尾部,结合使用连续截断变体的结合亲和力测定,AnkG-Nfasc结合的分子基础。我们确认AnkG与Nfasc中的FIGQY基序相互作用,我们确定了它们的高亲和力结合所需的另一个区域。我们的结构分析表明,ANK重复序列在AnkG内沟中形成4个疏水或亲水层,与必需的Nfasc残基协调相互作用,包括F1202、E1204和Y1212。此外,我们显示AnkG-Nfasc复合物的破坏消除了培养的小鼠海马神经元在AIS的Nfasc富集。最后,我们的结构和生化分析表明L1CAM中L1综合征相关突变,L1免疫球蛋白家族蛋白的成员,包括Nfasc,L1CAM,NrCAM,和CHL1,损害与锚蛋白的结合。一起来看,这些结果定义了AnkG-Nfasc复合物形成的潜在机制,并表明Nfasc的AnkG依赖聚类是AIS完整性所必需的。
The axon initial segment (AIS) has characteristically dense clustering of voltage-gated sodium channels (Nav), cell adhesion molecule Neurofascin 186 (Nfasc), and neuronal scaffold protein Ankyrin-G (AnkG) in neurons, which facilitates generation of an action potential and maintenance of axonal polarity. However, the mechanisms underlying AIS assembly, maintenance, and plasticity remain poorly understood. Here, we report the high-resolution crystal structure of the AnkG ankyrin repeat (ANK repeat) domain in complex with its binding site in the Nfasc cytoplasmic tail that shows, in conjunction with binding affinity assays with serial truncation variants, the molecular basis of AnkG-Nfasc binding. We confirm AnkG interacts with the FIGQY motif in Nfasc, and we identify another region required for their high affinity binding. Our structural analysis revealed that ANK repeats form 4 hydrophobic or hydrophilic layers in the AnkG inner groove that coordinate interactions with essential Nfasc residues, including F1202, E1204, and Y1212. Moreover, we show disruption of the AnkG-Nfasc complex abolishes Nfasc enrichment at the AIS in cultured mouse hippocampal neurons. Finally, our structural and biochemical analysis indicated that L1 syndrome-associated mutations in L1CAM, a member of the L1 immunoglobulin family proteins including Nfasc, L1CAM, NrCAM, and CHL1, compromise binding with ankyrins. Taken together, these results define the mechanisms underlying AnkG-Nfasc complex formation and show that AnkG-dependent clustering of Nfasc is required for AIS integrity.