AnkG-Neurofascin complex structure reveals binding mechanisms required for integrity of the AIS

The axon initial segment (AIS) has characteristically clustering of voltage-gated sodium channels (Nav), cell adhesion molecule Neurofascin (Nfasc), and neuronal scaffold protein Ankyrin-G (AnkG) in neurons, which facilitate generation of action potential and maintenance of axonal polarity. However, the mechanisms underlying AIS assembly and maintenance remain poorly understood. Here we report the high-resolution crystal structure of the AnkG in complex with a fragment from Nfasc cytoplasmic tail that shows, in conjunction with binding affinity assays, the molecular basis of AnkG-Nfasc binding. We confirm AnkG interacts with the FIGQY motif in Nfasc, and identify another region required for their high affinity binding. Structural analysis revealed that ANK repeats form four hydrophobic or hydrophilic layers in the AnkG inner groove that coordinate interactions with Nfasc. Moreover, disruption of the AnkG-Nfasc complex abolishes Nfasc enrichment at the AIS in hippocampal neurons. Finally, structural and biochemical analysis indicated that L1 syndrome-associated mutations in L1CAM compromise binding with ankyrins. These results define the mechanisms underlying AnkG-Nfasc complex formation and show that AnkG-dependent clustering of Nfasc is required for AIS integrity.