000170116 001__ 170116 000170116 005__ 20260318155255.0 000170116 0247_ $$2doi$$a10.1093/nar/gkag165 000170116 0248_ $$2sideral$$a148657 000170116 037__ $$aART-2026-148657 000170116 041__ $$aeng 000170116 100__ $$aRivero-García, Pablo 000170116 245__ $$aMultisite phosphorylation of the AML-linked C-terminal of nucleophosmin (NPM1) orchestrates protein stability, DNA binding and charge block-driven phase separation 000170116 260__ $$c2026 000170116 5060_ $$aAccess copy available to the general public$$fUnrestricted 000170116 5203_ $$aNucleophosmin (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. 000170116 536__ $$9info:eu-repo/grantAgreement/ES/CSIC/JAEINT24-EX-0171$$9info:eu-repo/grantAgreement/ES/MCIU/PID2024-157414NB-I00 000170116 540__ $$9info:eu-repo/semantics/openAccess$$aby-nc$$uhttps://creativecommons.org/licenses/by-nc/4.0/deed.es 000170116 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/publishedVersion 000170116 700__ $$aGiner-Arroyo, Rafael L 000170116 700__ $$aTamargo-Azpilicueta, Joaquín 000170116 700__ $$aTelfer, Abbey 000170116 700__ $$aFrezza , Elisa 000170116 700__ $$0(orcid)0000-0001-5702-4538$$aVelázquez-Campoy, Adrián$$uUniversidad de Zaragoza 000170116 700__ $$aDíaz-Moreno, Sofía 000170116 700__ $$aDe la Rosa, Miguel A. 000170116 700__ $$aDíaz-Moreno, Irene 000170116 7102_ $$11002$$2060$$aUniversidad de Zaragoza$$bDpto. Bioq.Biolog.Mol. Celular$$cÁrea Bioquímica y Biolog.Mole. 000170116 773__ $$g54, 5 (2026), 17 pp.$$pNucleic acids res.$$tNucleic Acids Research$$x0305-1048 000170116 8564_ $$s3440949$$uhttps://zaguan.unizar.es/record/170116/files/texto_completo.pdf$$yVersión publicada 000170116 8564_ $$s2545011$$uhttps://zaguan.unizar.es/record/170116/files/texto_completo.jpg?subformat=icon$$xicon$$yVersión publicada 000170116 909CO $$ooai:zaguan.unizar.es:170116$$particulos$$pdriver 000170116 951__ $$a2026-03-18-13:52:38 000170116 980__ $$aARTICLE