AbstractShank proteins are major scaffolds of the postsynaptic density of excitatory synapses. Mutations in SHANK genes are associated with autism and intellectual disability. The effects of missense mutations on Shank3 function, and therefore the pathomechanisms are unclear. Several missense mutations in SHANK3 affect the N-terminal region, consisting of the Shank/ProSAP N-terminal (SPN) domain and a set of Ankyrin (Ank) repeats. Here we identify a novel SHANK3 missense mutation (p.L270M) in the Ankyrin repeats in patients with an ADHD-like phenotype. We functionally analysed this and a series of other mutations, using biochemical and biophysical techniques. We observe two major effects: (1) a loss of binding to δ-catenin (e.g. in the p.L270M variant), and (2) interference with the intramolecular interaction between N-terminal SPN domain and the Ank repeats. This also interferes with binding to the α-subunit of the calcium-/calmodulin dependent kinase II (αCaMKII), and appears to be associated with a more severe neurodevelopmental pathology.