Substrate recognition principles for the PP2A-B55 protein phosphatase

Abstract

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.

Figure 1.. Helical motifs engage a conserved binding pocket on PP2A-B55

A) Schematic of pipeline to identify PP2A-B55 binding elements. B) Alignment of validated instances with helices in grey, residues contacting patch 1 in blue, patch 2 in yellow and patch 3 in red. The first 8 proteins on the list covers the proteins that was scanned by single Alanine mutagenesis and residues in italic was found to reduce binding by at least 50%. C) Model of the PP2A-B55-CDCA4 complex and the different patches in B55 indicated. D) AF models of the indicated proteins and their interaction with B55. Core residues interaction with the different patches in stick.

Figure 2.. Helical motifs can act as substrate specifiying elements

A) The indicated B55 mutants was purified and binding to IER2, PME1, CDCA4 and FAM122A determined and quantified.B) Quantifications of relative binding of proteins indicated to B55. C) Heat map illustrating the changes in binding pattern of the indicated proteins to the different B55 variants as determined by mass spectrometry. The scale is log2. D) The CDCA4 wt or mutant SERTA domain was fused to FOXO3 and binding to PP2A-B55 monitored by western blot. E) As D) but IPs probed with FOXO3 phosphospecific antibodies. F) Localisation of FOXO3 fusion proteins by live cell microscopy.

Figure 3.. Generation of a specific B55 inhibitor

A) Design pipeline for generating a specific and tight binder of B55 and test of the top designs by immunopurification. B) Model of the B55i bound to B55 and measured Kd and Ki indicated below. C) Coomassie stained gel of B55i and B55i CTRL purified from HeLa cells. D) B55 was affinity purified after incubation with either B55i or B55i CTRL peeptides and samples analysed by mass spectrometry. E) Mitotic duration in cells expressing B55i measured by timelapse microscopy. Scale bar is 5 μM. Right panel: Quantification of E). Shown is pooled data from three independent experiments. Each circle represents the timing of a single cell. Red line indicates the median time. Mann–Whitney U-test was applied. ns, not significant. ***P < 0.001.

Figure 4.. PP2A-B55 regulates NEXT complex function through binding RBM7

A) Stable HeLa cell lines expressing the indicated constructs and RNA levels of indicated NEXT substrates measured by RTqPCR. B) IP of RBM7 constructs and monitoring binding to B55. C) Mass spectrometry comparison of RBM7 WT and R143 with NEXT components in blue. D) Binding of RBM7 S136A and S136D to PP2A-B55 and table of affinities measured by SPR measurements of RBM7 peptides. E) Endogenous RBM7 was tagged with dTAG allowing rapid removal of RBM7 by adding dTAG and cells were complemented with the endicated RBM7 variants and the indicated RNAs were quantified by RTqPCR.