170116 20260318155255.0 doi 10.1093/nar/gkag165 sideral 148657 ART-2026-148657 eng Rivero-García, Pablo Multisite phosphorylation of the AML-linked C-terminal of nucleophosmin (NPM1) orchestrates protein stability, DNA binding and charge block-driven phase separation 2026 Nucleophosmin (NPM1) is a nucleolar protein commonly mutated in ~30% of newly diagnosed acute myeloid leukemia (AML) cases. These mutations occur in the terminal exon of the NPM1 gene, affecting the C-terminal DNA-binding domain of the protein and causing its delocalization to the cytoplasm—a hallmark of NPM1-mutated AML. NPM1 shuttling to the nucleoplasm is tightly regulated by posttranslational modifications, such as phosphorylation of Ser254, Ser260, and Tyr271 of the DNA-binding domain. However, the structural mechanisms underlying this process remain unclear. In this work, we show that Ser-to-Asp (S254D–S260D) and Tyr-to-pCMF (para-carboxymethyl phenylalanine) (Y271pCMF) phosphomimetic mutations induce significant structural and dynamical rearrangements, as well as drastic modifications in electrostatic surface potential. These changes compromise recognition of a G-quadruplex sequence from the c-MYC promoter by reducing DNA-binding affinity, reshape histone capturing dynamics, and fade charge segregation in the histone-binding domain. Combination of such substitutions in a triple phosphomimetic variant (S254D–S260D–Y271pCMF) further destabilizes the domain’s structure and triggers protein aggregation. Altogether, these findings suggest that phosphorylation of Ser254, Ser260, and Tyr271 of the C-end DNA-binding domain weakens both DNA affinity and charge block-driven liquid–liquid phase separation, offering a molecular explanation for the delocalization of NPM1 outside of the nucleolus. Access copy available to the general public Unrestricted info:eu-repo/grantAgreement/ES/CSIC/JAEINT24-EX-0171 info:eu-repo/grantAgreement/ES/MCIU/PID2024-157414NB-I00 info:eu-repo/semantics/openAccess by-nc https://creativecommons.org/licenses/by-nc/4.0/deed.es info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion Giner-Arroyo, Rafael L Tamargo-Azpilicueta, Joaquín Telfer, Abbey Frezza , Elisa Velázquez-Campoy, Adrián Universidad de Zaragoza (orcid)0000-0001-5702-4538 Díaz-Moreno, Sofía De la Rosa, Miguel A. Díaz-Moreno, Irene 1002 060 Universidad de Zaragoza Dpto. Bioq.Biolog.Mol. Celular Área Bioquímica y Biolog.Mole. 54, 5 (2026), 17 pp. Nucleic acids res. Nucleic Acids Research 0305-1048 3440949 http://zaguan.unizar.es/record/170116/files/texto_completo.pdf Versión publicada 2545011 http://zaguan.unizar.es/record/170116/files/texto_completo.jpg?subformat=icon icon Versión publicada oai:zaguan.unizar.es:170116 articulos driver 2026-03-18-13:52:38 ARTICLE