Small Molecule Activators of Protein Phosphatase 2A Exert Global Stabilizing Effects on the Scaffold PR65

Protein phosphatase 2A (PP2A), an important therapeutic target, comprises scaffold subunit PR65 composed of 15 HEAT (Huntingtin/Elongation/A-subunit/TOR1) repeats, a catalytic subunit, and one of many different regulatory subunits that enable binding to specific substrates. Recently, small molecule activators of PP2A (SMAPs) were identified, although their mechanisms of action have not yet been fully defined. Here, we explore the interaction of PR65 with two SMAPs, ATUX-8385 and the nonfunctional DBK-776, using single-molecule optical tweezers, ensemble methods, and computational analysis. In the absence of SMAP, PR65 shows multiple unfolding and refolding transitions, and the force–extension profiles are very heterogeneous with evidence of misfolding. Similar heterogeneity has been observed for the chemical-induced unfolding of tandem-repeat proteins like PR65, a consequence of the internal symmetry of the repeat architecture. In the presence of ATUX-8385, higher unfolding and refolding forces are observed throughout the structure and there is less misfolding, suggesting that ATUX-8385 acts like a pharmacological chaperone. In contrast, DBK-766-binding induces higher unfolding forces only for a few repeats of PR65, suggestive of a more localized effect; moreover, subsequent stretch–relax cycles show that PR65 is irreversibly locked in the unfolded state. Docking and molecular dynamics simulations provide insights into the distinctive responses of PR65 to mechanical stress in the presence of these two SMAPs: ATUX-8385 stably binds to a key site in the inner face of the PR65 structure, stabilizing a conformation predisposed to associate with the catalytic and regulatory subunits of PP2A. DBK-766, in contrast, exhibits a weaker binding to the outer face of PR65 and elicits relatively large conformational fluctuations in PR65 when bound to the compact form.