Structural insight of PP2A inhibitor proteins and their interaction with PP2A Aand B56-subunits

Abstract

Protein phosphatase 2A (PP2A), the principal Serine/Threonine phosphatase, functions as a tumor suppressor. In most of the cancers, the PP2A tumor suppressor activity is inhibited either genetically or by the overexpression of PP2A inhibitor proteins. It is therapeutically tempting idea to reactivate inhibited PP2A by small molecule modulators, For that, a better understanding of the structural and molecular framework of PP2A inhibition is required. In this study, we have characterized the structural properties of PP2A inhibitor proteins ARPP- 19, ARPP-16, and ENSA, and their interaction with A- and B56-subunits of PP2A by combining NMR spectroscopy with SAXS and microscale thermophoresis. The results reveal that both ENSA and ARPP proteins are intrinsically disordered, but not completely random coil as they have three regions having the propensity to form transient α-helical structures. Both ARPPs and ENSA were observed to interact with PP2A A-subunit with modest affinity, whereas, the interaction with B56-subunit is weak and transient. When ARPP and ENSA proteins are phosphorylated by Gwl/MAST3 kinase they inhibit PP2A during mitosis. The phosphomimetic mutants resembling phosphorylation of ARPPs showed increased affinity towards both A- and B56- subunits, while the corresponding phosphomimetic mutants of ENSA failed to bind to all other B56 subunits except B56α. Two distinct interaction modes of ARPPs and ENSA with the PP2A A-subunit were identified. In ARPPs, a second transient α-helix including its flanking region forms an A-subunit binding motif, whereas ENSA interacts with A-subunit using an extended region comprising all three transient α-helical regions. Together, these studies suggest that intrinsically disordered ARPPs and ENSA bind to PP2A transiently using preformed structural elements. Altogether, our results provide a crucial step towards the understanding the molecular bases behind PP2A inhibition, which provide a foundation for the development of novel and clinically feasible PP2A targeted therapies. Keywords: ARPP-16/ARPP-19; ENSA; Intrinsically Disordered proteins; NMR, Protein Phosphatase 2A, SAXS.