System and MD simulations-

The PR65 structure resolved for the trimeric PP2A, deposited in the Protein Data Bank (PDB: 6NTS)³ was used as starting conformation in our simulations. PR65 is in its compact form in the trimer. The structure was simulated in TIP3 explicit water having 25 Å of water padding in all directions. Thus, providing at least 50 Å of water between PR65 and its periodic images. Ions were added to neutralize the systems and ion concentrations were set to 150 mM NACl. System size was ~ 164,147 atoms for WT PR65 simulations. All system preparations were performed in VMD and all MD simulations were performed in NAMD3⁵¹ using the CHARMM36 all-atom additive protein force field⁵². A time step of 2 fs was used in the simulations. Temperature was kept constant at 310 K via Langevin dynamics using a damping coefficient of 1 ps^{− 1}. The pressure was kept at 1 atm using the Langevin Nosé–Hoover method with an oscillation period of 100 fs and a damping time scale of 50 fs. A cut-off distance of 12 Å was applied for van der Waals interactions. To calculate long-range electrostatic interactions, the particle-mesh Ewald method was used. PR65 mutants were generated by introducing single point mutations using the mutator plugin of VMD. PR65 mutants were simulated following the steps indicated above. Two rounds of minimization and equilibration simulations were performed prior to each production run. First, the protein was maintained in a fixed structure and the system was subjected to 10,000 energy minimization steps, which was followed by 1 ns of stabilization to equilibrate the solvent around the protein. Subsequently, a second round of minimization-equilibration was performed, where each system underwent an additional 10,000 step minimization, this time without any restrictions, which was subsequently followed by 4 ns of stabilization using harmonic constraints (of 1 kcal/mol/Ų) on C^α-atoms only. Following these simulations, constraints were completely removed, and the system went through another round of 4 ns equilibration. Upon completion of this second round of minimization-equilibration, production runs were performed.

Conformations were sampled every 0.1 ns in the MD simulations and used in RMSD, RMSF, and PCA calculations. Thus, when combining the three sets of simulations for each of the WT and mutant systems, we obtained an ensemble of 19620 snapshots from a total of 3×654 ns = 1962 ns of MD simulations. Cumulatively MD simulations of 13.734 μs were evaluated. We performed all calculations using our custom analysis codes, executed in VMD and MATLAB, which also utilized some of their built-in functions.