MassIVE MSV000099747

Complete Public PXD070300

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

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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. [doi:10.25345/C5JQ0T78N] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: intrinsically disordered protein ; de-phosphorylation ; protein phosphatase ; calcineurin ; clinical missense variant ; DatasetType:Proteomics

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Principal Investigators:
(in alphabetical order) 		Arminja Kettenbach, The Geisel School of Medicine at Dartmouth, United States
Submitting User: Arminja
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