Substrate 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 modelling 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 complex by binding to an α-helical recruitment module in RBM7. Collectively, our findings provide a framework for the understanding and interrogation of PP2A-B55 in health and disease.