biostudies-literatureKeskitalo SInstitut National de la Santé et de la Recherche Médicale (National Institute of Health and Medical Research)Agence Nationale de la Recherche (French National Research Agency)586https://www.ebi.ac.uk/biostudies/studies/S-EPMC12808100biostudies-literatureUnknown17(1)Short linear motifs (SLiMs) in disordered protein regions direct numerous protein-protein interactions, yet most remain uncharacterized. The Asn-Pro-Phe (NPF) motif is a well-known EH-domain ligand implicated in endocytosis, but here we reveal that the non-catalytic subunit of human DNA polymerase ε (POLE2) also serves as a general NPF-motif receptor. Using a quantitative "native holdup" assay, we find that POLE2 selectively binds diverse NPF-containing peptides, including canonical EH-domain ligands (e.g., SYNJ1) and previously uncharacterized motifs. Biochemical measurements and mutational analyses show that NPF motifs interact with a shallow pocket near the POLE2 C-terminus, and AlphaFold predictions confirm key roles for Y513, E520, and S522 in motif coordination. Proteome-scale affiniNature communicationsThe non-catalytic DNA polymerase ε subunit is an NPF motif recognition protein.PMC12808100ANR-24-ERCS-0002ATIP-AvenirZambo BGogl GVarjosalo MDeutsch NKeskitalo SMalaymar Pinar DTuhkala ADavey NSalokas KDosztanyi ZTurunen TfalseThe non-catalytic DNA polymerase ε subunit is an NPF motif recognition protein.Short linear motifs (SLiMs) in disordered protein regions direct numerous protein-protein interactions, yet most remain uncharacterized. The Asn-Pro-Phe (NPF) motif is a well-known EH-domain ligand implicated in endocytosis, but here we reveal that the non-catalytic subunit of human DNA polymerase ε (POLE2) also serves as a general NPF-motif receptor. Using a quantitative "native holdup" assay, we find that POLE2 selectively binds diverse NPF-containing peptides, including canonical EH-domain ligands (e.g., SYNJ1) and previously uncharacterized motifs. Biochemical measurements and mutational analyses show that NPF motifs interact with a shallow pocket near the POLE2 C-terminus, and AlphaFold predictions confirm key roles for Y513, E520, and S522 in motif coordination. Proteome-scale affini2025-01-01T00:00:00Z2025 Dec2026-06-06T15:39:39.112Z2026-06-02T03:09:05.729ZS-EPMC128081004139076910.1038/s41467-025-67284-5