Structural Interactions of Ankyrin B with NrCAM and β ₂ Spectrin

Ank2 is a high confidence autism spectrum disorder (ASD) gene encoding the spectrin-actin scaffold protein Ankyrin B (AnkB). The 220 kDal isoform of AnkB has multiple functions including developmental spine pruning through L1 family cell adhesion molecules (L1-CAMs) and class 3 Semaphorins on dendrites of pyramidal neurons to achieve an appropriate excitatory balance in the neocortex. Molecular modeling employing AlphaFold was used to predict the structure and interactions of AnkB with the cytoplasmic domain of Neuron-glial Related L1-CAM (NrCAM), and with β2-Spectrin. The validity of the models was assessed by analyzing protein-protein interactions by co-immunoprecipitation from HEK293 cell lysates after mutating key residues in AnkB predicted to impair these associations. Results revealed a pocket with critical residues in the AnkB membrane-binding domain that engages NrCAM at the conserved cytoplasmic motif FIGQY. Alphafold modeling of the AnkB/β2-Spectrin complex also identified key interactions between the AnkB spectrin-binding domain and β2-Spectrin repeats 14-15. Selected ASD-linked mutations in AnkB predicted to impact binding to NrCAM or β2-Spectrin were then assayed for protein interactions. Maternally inherited ASD missense mutations AnkB A368G located in the NrCAM binding pocket and AnkB R977Q in the Zu51 subdomain disrupted associations with NrCAM and β2-Spectrin, respectively. Moreover, AnkB A368G impaired the neuronal function of 220 kDal AnkB in Semaphorin 3F-induced spine pruning in mouse cortical neuron cultures. These new findings provide structural insights into the L1-CAM/AnkB complex and the molecular basis of ASD etiology associated with AnkB missense mutations.