# Small Molecule Activator of Phosphatase PP2A Remodels Scaffold PR65 Structural Dynamics To Promote Holoenzyme Assembly

## TL;DR

This study explores how small molecules activate a protein phosphatase by altering the structure of its scaffold subunit to promote enzyme assembly.

## Contribution

The study reveals how SMAPs bind to PR65 and influence its structural dynamics to facilitate holoenzyme assembly.

## Key findings

- SMAPs bind stably to S2 and S4 sites on PR65, but not to S1.
- Binding to S2 and S4 stabilizes an extended PR65 conformation that promotes holoenzyme assembly.
- Mutations at S2 and S4 destabilize SMAP binding to PR65.

## Abstract

Small molecule activators of protein phosphatase 2A (PP2A),
hereafter
SMAPs, have attracted substantial interest for their potential to
inhibit cancer cell proliferation by targeting PR65, the scaffold
subunit of the PP2A heterotrimer. PR65 is a uniquely flexible and
stable molecule composed of 15 tandem HEAT (Huntingtin, Elongation
factor 3–PP2A–TOR1) repeats. We characterized the binding
sites and interactions of two SMAPs ATUX-8385 and DT-061 with PR65
and evaluated the effects on PR65 structural dynamics using docking
and molecular dynamics simulations. We initiated SMAP-bound PR65 simulations
starting from two binding sites: S1, determined by cryo-electron microscopy
for DT-061 bound to PP2A, on the inner helices of the HEAT repeats
2 and 3 (2i and 3i); and S2, predicted by docking
of ATUX-8385 onto PR65, on 4i and 5i and outer
helices 5o and 6o consistent with footprinting
experiments. S2 proved to be a stable site for both SMAPs when simulations
were initiated at S2. However, neither DT-061 nor ATUX-8385 demonstrated
stable binding to S1. DT-061 rapidly dissociated from S1 to settle
instead at a neighboring site S4 overlapping with our previously identified
S3 for PR65 in extended form, suggesting that binding to S1 may be
a two-step process: an initial binding to PR65 alone, either to S3/S4
or S2, followed by movement to S3/S4, and then an induced relocation
to S1 upon complexation with the regulatory and catalytic subunits.
Targeted in silico mutagenesis showed that mutations at S2 and S4
destabilized binding of SMAP to PR65 (subunit). Heterotrimeric PP2A
simulations showed that S3 and S4 bindings were not persistent upon
complexation. Together, these results corroborate our findings. Furthermore,
this preferentially stabilized a relatively extended PR65 conformation
that would accommodate, if not promote, the assembly of the catalytic
and regulatory subunits to prompt the activation of the trimeric phosphatase.