Direct observation of small molecule activator binding to single PR65 protein

The reactivation of heterotrimeric protein phosphatase 2A (PP2A) through small molecule activators is of interest to therapeutic intervention due to its dysregulation, which is linked to chronic conditions. This study focuses on the PP2A scaffold subunit PR65 and a small molecule activator, ATUX-8385, designed to bind directly to this subunit. Using a label-free single-molecule approach with nanoaperture optical tweezers (NOT), we quantify its binding, obtaining a dissociation constant of 13.6 ± 2.5 μM, consistent with ensemble fluorescence anisotropy results but challenging to achieve with other methods due to low affinity. Single-molecule NOT measurements reveal that binding increases optical scattering, indicating PR65 elongation. This interpretation is supported by all-atom molecular dynamics simulations showing PR65 adopts more extended conformations upon binding. This work highlights NOT's utility in quantifying binding kinetics and structural impact, offering insights valuable for drug discovery.