doi:10.1093/nar/gkag165engRivero-García, PabloGiner-Arroyo, Rafael LTamargo-Azpilicueta, JoaquínTelfer, AbbeyFrezza , ElisaVelázquez-Campoy, AdriánDíaz-Moreno, SofíaDe la Rosa, Miguel A.Díaz-Moreno, IreneMultisite phosphorylation of the AML-linked C-terminal of nucleophosmin (NPM1) orchestrates protein stability, DNA binding and charge block-driven phase separationART-2026-148657Nucleophosmin (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.2026http://zaguan.unizar.es/record/17011610.1093/nar/gkag165http://zaguan.unizar.es/record/170116oai:zaguan.unizar.es:170116info:eu-repo/grantAgreement/ES/CSIC/JAEINT24-EX-0171info:eu-repo/grantAgreement/ES/MCIU/PID2024-157414NB-I00Nucleic Acids Research 54, 5 (2026), 17 pp.by-nchttps://creativecommons.org/licenses/by-nc/4.0/deed.esinfo:eu-repo/semantics/openAccess