Prediction of PP2A Holoenzymes Guided By the Structural Characterization of Methylation Independent PP2A Assembly
Authors: Wachter Franziska , Nowak Radoslaw , Fischer Eric 
Published: December 2023

Introduction:
The dysregulation of phosphorylation-dependent signaling is a hallmark of leukemogenesis. Kinase inhibitors have revolutionized the outcomes of patients with chronic myeloid leukemia. An orthogonal, but less explored way to target phosphorylation pathways is to activate phosphatases, such as the ubiquitously expressed protein- phosphatase 2A (PP2A). PP2A is an essential serine/threonine phosphatase that regulates various cellular processes, including cell growth, division, and differentiation. Dysregulation of PP2A has been linked to leukemia. PP2A is a modular multi-subunit enzyme: One scaffold subunit (A) binds to a catalytic subunit (C) to form a core AC heterodimer, which together with one of many regulatory (B) subunits forms the active enzyme. The regulatory (B) subunits are classified into 4 distinct subfamilies without significant homology. The combinatorial assembly of these subunits generates a wide range of PP2A complexes, resulting in diverse substrate specificity and subcellular localization. The detailed mechanism of PP2A regulation remains elusive and reports about a governing role of methylation of the carboxy-terminal Leucin of PP2A C are conflicting. Understanding the underlying molecular regulation of PP2A activity is of significant interest for future therapeutic development for acute myeloid leukemia.
Methods:
Recombinant PP2A was expressed in insect cells, and purified by affinity purification, anion exchange chromatography and size exclusion chromatography (SEC). Extensive quality control, including liquid chromatography-mass spectrometry, SDS-PAGE gel and fast protein liquid chromatography was performed. The degree of methylation of PP2A C Leucine 309 was confirmed by mass spectrometry. We validated protein stability with differential scanning fluorimetry. Pull-down assays and size exclusion chromatography characterized complex formation. A calorimetric assay using p-nitrophenyl phosphate as a substrate functionally assessed PP2A in vitro. Structural information was obtained by X-ray crystallography and AlphaFold Multimer.
Results:
Using in vitro biochemistry, including pull down and size exclusion chromatography, we demonstrate that methylation of the carboxy terminus of the PP2A C subunit is dispensable for PP2A assembly in vitro. Absence of methylation of Leucine 309 was confirmed by mass-spectrometry. We show by SEC that instead PP2A complex formation is dependent on B subunit concentration. Carboxy-terminal methylation of the PP2A C subunit was also not required for functional activity in a calorimetric assay using p-nitrophenyl phosphate as a substrate. PP2A formed spontaneously in vitro and was enzymatically active independent of methylation of Leucine 309. To corroborate these findings, we determined the X-ray crystal structure of the PP2A:PPP2B56E:PPP2C complex to 2.7 Å resolution with the unmethylated Leucine 309. Interestingly our experimental structure superimposed with an AlphaFold Multimer model of the PP2A trimer. Building on this finding, we predicted the models of all 64 possible PP2A complexes providing a framework for further study and structural analysis of PP2A complexes and their activation. These studies provide key information for the structural design and development of novel drugs for hematologic malignancies by targeting of selected PP2A complexes and clarified the role of Leucine 309 methylation in PP2A activation.
Conclusion:
Methylation of the carboxy-terminal Leucine 309 of the PP2A C subunitis dispensable for PP2A activity in vitro. Integrative structural biology studies, combining biochemistry, X-ray crystallography and AlphaFold Multimer provide a comprehensive map of PP2A complexes. These studies have the potential to accelerate the drug discovery process and unlock PP2A activation as a new therapeutic modality for leukemia.