biostudies-literatureKruse TWellcome TrustNIGMS NIH HHSeadp5491https://www.ebi.ac.uk/biostudies/studies/S-EPMC11446282biostudies-literatureUnknown10(40)The PP2A-B55 phosphatase regulates a plethora of signaling pathways throughout eukaryotes. How PP2A-B55 selects its substrates presents a severe knowledge gap. By integrating AlphaFold modeling with comprehensive high-resolution mutational scanning, we show that α helices in substrates bind B55 through an evolutionary conserved mechanism. Despite a large diversity in sequence and composition, these α helices share key amino acid determinants that engage discrete hydrophobic and electrostatic patches. Using deep learning protein design, we generate a specific and potent competitive peptide inhibitor of PP2A-B55 substrate interactions. With this inhibitor, we uncover that PP2A-B55 regulates the nuclear exosome targeting (NEXT) complex by binding to an α-helical recruitment module in the RNA Science advancesSubstrate recognition principles for the PP2A-B55 protein phosphatase.PMC11446282R35 GM119455Jensen THVarga JKHein JBMendez BLBenavides-Puy INilsson JJeyaprakash AASotelo-Parrilla PKettenbach ANChan CBGarland WWeisser MBSchueler-Furman OKruse TMcEwan BCGarvanska DHfalseSubstrate recognition principles for the PP2A-B55 protein phosphatase.The PP2A-B55 phosphatase regulates a plethora of signaling pathways throughout eukaryotes. How PP2A-B55 selects its substrates presents a severe knowledge gap. By integrating AlphaFold modeling with comprehensive high-resolution mutational scanning, we show that α helices in substrates bind B55 through an evolutionary conserved mechanism. Despite a large diversity in sequence and composition, these α helices share key amino acid determinants that engage discrete hydrophobic and electrostatic patches. Using deep learning protein design, we generate a specific and potent competitive peptide inhibitor of PP2A-B55 substrate interactions. With this inhibitor, we uncover that PP2A-B55 regulates the nuclear exosome targeting (NEXT) complex by binding to an α-helical recruitment module in the RNA 2024-01-01T00:00:00Z2024 Oct2025-04-18T15:57:14.96Z2025-04-07T02:52:53.03ZS-EPMC114462823935675810.1126/sciadv.adp5491