The clinical missense variant E282K in PPP3CA/calcineurin shifts substrate dephosphorylation by altering active site recruitment

Recently, de novo heterozygous variants of Calcineurin (CN) were reported as the cause of a neurodevelopmental disorder that presents with epileptic encephalopathy and dysmorphism (DEE91), with the largest group of patients harboring the CN missense mutation E282K (glutamate → lysine). Here, we use molecular and cellular techniques to define how this mutation alters CN activity. We discover that basophilic substrates use an arginine residue to bind to CN via an acidic substrate recruitment pocket adjacent to the CN active site, the E282 pocket. Furthermore, we show that basic residues in the i-1 position of the substrate relative to the substrate phosphosite enhance CN-mediated dephosphorylation. While the CN_E282K structure shows that the overall conformation is unchanged, the E282 pocket transforms from acidic to basic, with pocket access blocked by the formation of a E282K-E237 salt bridge. Finally, in vitro assays and in cell phosphoproteomics show that CN_E282K shifts CN substrate dephosphorylation profiles from basic to acidic, thereby altering CN-mediated dephosphorylation signaling. Together, these data define the molecular impact of the CN_E282K variant in cells and development, providing a key step for developing strategies to treat this disorder and its accompanying complications.