Kruse, T.; Garvanska, D.; Varga, J. K.; Garland, W.; McEwan, B.; Hein, J.; Weisser, M. B.; Brandies Puy, I.; Chan, C.; Parrila, P. S.; Lopez-Mendez, B.; Jeyaprakash, A. A.; Schueler-Furman, O.; Jensen, T. H.; Kettenbach, A.; Nilsson, J.

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 alpha helices in substrates bind B55 through an evolutionary conserved mechanism. Despite a large diversity in sequence and composition, these alpha 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 a helical recruitment module in RBM7. Collectively, our findings provide a framework for the understanding and interrogation of PP2A-B55 in health and disease.