Protein phosphatase 2A (PP2A) is a heterotrimeric enzyme whose assembly and activity are orchestrated by the flexible scaffold PR65, a 15 HEAT repeat protein capable of compact and extended conformations. Small-molecule activators of PP2A (SMAPs), including DT-061 and ATUX-8385, have emerged as promising therapeutic modulators, yet the structural basis of their action on PR65 remains unresolved. Here, we combined molecular docking with extensive all-atom molecular dynamics (MD) simulations (∼13 μs) to map SMAP binding sites and their effects on PR65 conformational dynamics. Simulations tested two candidate sites: S1, the cryo-EM pocket for DT-061 between helices 2 i -3 i , and S2, a docking-predicted site spanning helices 4 i -5 i and 5 o -6 o . S2 proved to be a high-affinity site for both SMAPs, stabilized by Y154, R166, F191, and N199. By contrast, SMAPs bound at S1 on PR65 alone were unstable; both ligands dissociated rapidly, with DT-061 consistently relocating to a distinct site, S4 (helices 5 i -7 i ), overlapping a previously identified S3 pocket in extended PR65. At S4, anchoring residues L221 and R257 stabilized DT-061 binding. These data support a sequential mechanism in which SMAPs first capture PR65 at S2 or S4, depending on conformational state, then transition through S3 into the S1 pocket that stabilizes only in the context of holoenzyme assembly. Binding at S3 or S4 biased PR65 toward extended states, favoring recruitment of catalytic and regulatory subunits. Our findings reveal how SMAPs remodel PR65 to stabilize conformations that promote trimer formation and PP2A activation, providing a structural rationale for their therapeutic mechanism.
Yilmaz et al. (Sun,) studied this question.
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