Multisite phosphorylation of the AML-linked C-terminal of nucleophosmin (NPM1) orchestrates protein stability, DNA binding and charge block-driven phase separation

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Abstract
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.
Funding Information
  • Spanish State Research Agency
  • Ministry of Science, Innovation and Universities (PID2024-157414NB-I00)
  • Ministry of Science, Innovation and Universities (PRE2019-091404)
  • Ministry of Science, Innovation and Universities (FPU22/01094)
  • Ministry of Science, Innovation and Universities (FPU24/00691)
  • Ramón Areces Foundation (LEUCYTO-FRA2025-MAR)
  • Spanish Ministry of Education, Vocational Training and Sports (23CO1/008490)
  • Spanish Council of Scientific Research (JAEINT24_EX_0171)

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