Structural Interactions of Ankyrin B with NrCAM and β ₂ Spectrin

Area of Science:

• Neuroscience
• Molecular Biology
• Genetics

Background:

• Ankyrin B (AnkB) is encoded by the Ank2 gene, a high-confidence autism spectrum disorder (ASD) gene.
• The 220 kDal AnkB isoform regulates dendritic spine pruning via L1-CAMs and Semaphorins, crucial for neocortical excitatory balance.

Purpose of the Study:

• To structurally model Ankyrin B (AnkB) interactions with Neuron-glial Related L1-CAM (NrCAM) and β2-Spectrin using AlphaFold.
• To validate these models through mutagenesis and co-immunoprecipitation assays.
• To investigate the impact of ASD-linked AnkB mutations on protein interactions and neuronal function.

Main Methods:

• AlphaFold molecular modeling to predict AnkB complex structures with NrCAM and β2-Spectrin.
• Site-directed mutagenesis of AnkB to disrupt predicted interaction sites.
• Co-immunoprecipitation assays in HEK293 cells to assess protein-protein interactions.
• Assessment of AnkB A368G mutation's effect on Semaphorin 3F-induced spine pruning in mouse cortical neurons.

Main Results:

• AlphaFold models revealed critical AnkB residues for NrCAM binding (FIGQY motif) and β2-Spectrin interaction (repeats 14-15).
• ASD-linked mutations AnkB A368G and AnkB R977Q impaired binding to NrCAM and β2-Spectrin, respectively.
• AnkB A368G mutation disrupted Semaphorin 3F-induced spine pruning in cultured mouse neurons.

Conclusions:

• Structural insights into the L1-CAM/AnkB complex and β2-Spectrin interactions are provided.
• ASD-associated AnkB missense mutations can disrupt critical protein interactions.
• These disruptions offer a molecular basis for ASD etiology linked to Ank2 mutations.