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The clinical missense variant E282K in PPP3CA globally shifts substrate dephosphorylation profiles by altering active site recruitment

ABSTRACT: Whole exome sequencing led to the discovery that de novo heterozygous variants of Calcineurin are present in patients with a distinct neurodevelopmental disorder that presents with epileptic encephalopathy and dysmorphism. The largest group of patients harbor the CN missense mutation E282K. Defining how this mutation alters CN activity in vitro and in cells is essential for developing novel strategies to treat this disorder and its accompanying complications. Here, by integrating x-ray crystallography, NMR spectroscopy, biophysics and substrate-based activity assays, we identified a novel phosphosite interaction pocket on CN that is critical for enhancing the dephosphorylation of CN substrates. We then showed that this enhancement is due to a specific interaction between CN E282 and an arginine residue that immediately precedes the substrate phosphosite, and, more importantly, that this enhancement is abolished in the E282K variant. Finally, using a cellular phosphoproteomics approach, we show that the CN E282K variant shifts CN substrate dephosphorylation profiles, thereby globally altering signaling. Together, these data provide the key mechanistic insights to understand the impact of the CN E282K variant in cells and development and to identify disrupted signaling pathways that may lead to treatments for this disorder.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Human

SUBMITTER: Arminja Kettenbach

PROVIDER: MSV000098060 | MassIVE | Sat May 31 08:49:00 BST 2025

SECONDARY ACCESSION(S): PXD064473

REPOSITORIES: MassIVE

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

Project description: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 to lysine). Here, we use molecular and cellular techniques to define how this mutation alters CN activity. We discovered that basophilic substrates use an arginine residue to bind to CN via a newly discovered 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 CNE282K structure showed that the overall conformation is unchanged, the E282 pocket transformed from an acidic to basic, with pocket access blocked by the formation of a E282K-E237 salt bridge. Finally in vitro assays and in cell phosphoproteomics showed that CNE282K universally shifts CN substrate dephosphorylation profiles from basic to acidic, thereby globally altering CN-mediated dephosphorylation signaling. Together, these data define the molecular impact of the CNE282K variant in cells and development, providing a first step for developing new strategies to treat this disorder and its accompanying complications.

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