BPS2026 – The clinical missense variant E282K in PPP3CA/calcineurin shifts substrate dephosphorylation profiles by altering active site recruitment

Calcineurin (CN) is a calcium-activated serine/threonine phosphatase (PPP) that is involved in key cellular processes including development, synaptic plasticity, and memory formation. Consequently, the disruption of CN signaling is associated with multiple developmental and neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease. Unlike kinases, which recognize substrates using kinase-specific phosphosite motifs, PPPs recruit substrates using short linear motifs that are distal from the target phosphosite, with CN recruiting substrates using a PxIxIT and/or LxVP SLiM. However, how pockets adjacent to the CN active site influence CN activity is still an open question. Here, we identified a novel substrate recruitment pocket adjacent to the CN active site by determining the crystal structure of a CN-substrate trapped complex, in which the thiophosphorylated substrate (TAK1) was bound and trapped at the CN active site (using a catalytically inactive CN variant). The structure revealed that an arginine residue at the i-1 position of the phosphosite is crucial for substrate recruitment and dephosphorylation, as it binds an acidic pocket defined by CN residues E237 and E282. Notably, a de novo heterozygous variant of CN, E282K, was recently identified to be the most prevalent missense mutation in patients with a newly identified neurodevelopmental disorder that presents with epileptic encephalopathy and dysmorphism. Using structure-based biochemical, biophysical, and proteomic studies, we then showed that the recruitment of CN substrates with an Arg in the i-1 position is disrupted in CN E282K, and, furthermore, that this variant shifts signaling outcomes and impairs CN substrate dephosphorylation. Together, these data define the molecular impact of the CN E282K variant in cells and development, providing a first step for developing new strategies to treat this disorder and its accompanying complications.