Abstract
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 CNE282K 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 CNE282K shifts CN substrate dephosphorylation profiles from basic to acidic, thereby altering CN-mediated dephosphorylation signaling. Together, these data define the molecular impact of the CNE282K variant in cells and development, providing a key step for developing strategies to treat this disorder and its accompanying complications.
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Data availability
The NMR data generated in this study have been deposited in the BioMagResBank database under accession code BMRB 51895 (Sequence-specific backbone assignment of the intrinsically disordered C-terminal tail of TAK1) and 52982 (Sequence-specific backbone assignment of the cAMP-dependent protein kinase type II-alpha regulatory subunit disordered region (aa 44–103)). The atomic coordinates and structure factors generated in this study have been deposited in the PDB database under accession code 9NXE, 9NXF, 9NXN, 4F0Z, 6NUC and 5SVE. The raw mass spectrometry data is available at ProteomeXchange PXD064473 and PXD070300 and MassIVE MSV000098060 [https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=3714453b38b844cbbd8d5a35446320a9] and MSV000099747 [https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=b9958cb55d234bcebf59af63e7a81442]. ITC/SPR and enzymatic data generated in this study are provided in the Supplementary Information and/or Source Data file, which is available at Figshare (https://doi.org/10.6084/m9.figshare.29250827) and as a Source Data file. Source data are provided with this paper.
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Acknowledgements
We thank the PPP3CA Hope Foundation (https://www.ppp3ca.org/) for fruitful discussions. We acknowledge initial efforts to analyze the interaction of TAK1 with CN by Dr. Sarah Sheftic and help with the CN:RII crystallization by Dr. Thomas Moon. The authors would like to thank staff members at the Stanford Synchrotron Radiation Light source (SSRL), SLAC National Accelerator Laboratory for access to X-ray beamlines. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. This work was supported by grant R01GM144483 from the National Institute of General Medicine and R01NS124666 from the National Institute of Neurological Disorders and Stroke to W.P. and grant R01GM098482 from the National Institute of General Medicine to R.P. In part, this research was supported by the PPP3CA Hope Foundation Inc (to W.P.).
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R.P. and W.P. developed the concept. K.T.S., T.P. and L.E.S.F.M. expressed and purified all proteins. T.P. and L.E.S.F.M. performed NMR experiments. L.E.S.F.M. and K.T.S. performed ITC experiments. K.T.S. crystallized CND90A:tpTAK1 and determined the structures of the CND90A:tpTAK1 and CN:RII complexes. TP crystalized and determined the structure of CNE282K:NHE1. H.T.N., G.P. and A.N.K. performed phospho-library and phosphoproteomics experiments. H.T.N., T.P. and A.N.K. analyzed MS data. M.L.D. interpreted CN activity data. R.P., K.T.S., T.P. and W.P. wrote the manuscript with comments and inputs from all co-authors.
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Shirakawa, K.T., Parikh, T., Machado, L.E. et al. The clinical missense variant E282K in PPP3CA/calcineurin shifts substrate dephosphorylation by altering active site recruitment. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69535-5
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DOI: https://doi.org/10.1038/s41467-026-69535-5
