Deciphering the mechanistic basis for the pathological effect of the Gαo E246K mutation in neurodevelopmental disorder

Mutations in the GNAO1 gene, which encodes for Go, a major neuronal G protein, are associated with neurodevelopmental disorders, epilepsy, and movement disorders. We identified and characterized a spontaneous heterozygous GNAO1 E246K mutation in an Israeli female infant with complex developmental delays and substantial motor difficulties. This mutation has been reported in other cases as a prevalent pathogenic mutation in patients with motor dysfunction and a broad range of neurological outcomes. To investigate the molecular and functional consequences of the Go E246K mutation, we employed structural modeling and analysis, biochemical assays, mass spectrometry-based proteomics, and cellular functional assays. We show that this mutation does not affect nucleotide binding, nor basal or RGS- accelerated GTP hydrolysis. Despite the E246 position located within a predicted effector binding region, proteomics analysis did not identify any new cellular partners. Instead, we demonstrate that the E246K mutation disrupts the Go regulatory GTPase cycle by directly impairing G{beta}{gamma} dissociation. This impairment overrides the presence of wild-type Go, explaining the dominant effect of the severe neurogenetic phenotype in the heterozygous background. These findings establish a new molecular mechanism for a GNAO1 mutation with dominant-negative effects on the GTPase regulatory cycle. The insights gained from studying this mechanism of action provide a basis for developing specific and personalized therapeutic strategies based on the outcome of a missense mutation in GNAO1.