Patient‐Derived Variants Define Constraints for Ligand Binding at the <scp>PDZ</scp> Domain of <scp>CASK</scp>

Genetic variants in the X-chromosomal gene coding for the calcium-/calmodulin-dependent serine protein kinase (CASK) are associated with a neurodevelopmental disorder. CASK is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins. It acts as a scaffold at presynaptic sites, as a regulator of the transport of glutamate receptors, and as a transcriptional regulator. The PDZ domain of CASK has been reported to bind to presynaptic cell adhesion molecules such as Neurexin1-3, CNTNAP2, SynCAM and SALM1. Structural analyses of related MAGUKs indicate that the canonical SH3 and GK domains combine with the PDZ domain to form the so-called PSG supramodule. Conserved aromatic residues (Y723 and W914) flanking the GK domain contribute to the formation of a dimeric structure of two PSG modules, which is required for high-affinity binding to the type 2 PDZ ligand motif of, for example, Neurexin. Here we identify previously uncharacterized patient variants in the SH3 domain of CASK (I672V; P673L), which alter the intermolecular binding pocket for Y723. Both variants interfere with the binding of Neurexin-1β, in a manner similar to the previously reported Y723C variant. Intriguingly, binding to the type 1 PDZ ligand of the cell adhesion molecule SALM1 is not altered. Using a set of highly selective patient variants, we show that the binding of SALM1 to CASK is actually not mediated by the CASK PDZ domain or the PSG supramodule, but depends on other type 1 PDZ domain-containing proteins such as SAP97 and Veli, which associate with CASK through its L27 domains. Our data underline the relevance of an intact PSG tandem of CASK for human health.