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Phase separation of proteins plays a critical role in cellular organization. How phase-separated protein condensates underpin biological function and how condensates achieve specificity remain elusive. We investigated the phase separation of MUT-16, a scaffold protein in Mutator foci, and its role in recruiting the client protein MUT-8, a key component in RNA silencing in C. elegans. We employed a multiscale approach that combined coarse-grained (residue-level CALVADOS2 and near-atomistic Martini3) and atomistic simulations. Simulations across different resolutions provide a consistent perspective on how MUT-16 condensates recruit MUT-8, enabling the fine-tuning of chemical details while balancing the computational cost. Both coarse-grained models (CALVADOS2 and Martini3) predicted the relative phase separation propensities of MUT-16's disordered regions, which we confirmed through in vitro experiments. Simulations also identified key sequence features and residues driving phase separation while revealing differences in residue interaction propensities between CALVADOS2 and Martini3. Furthermore, Martini3 and 350 microsecond atomistic simulations on Folding@Home of MUT-8's N-terminal prion-like domain with the MUT-16 M8BR cluster highlighted the importance of cation-pi interactions between Tyr residues of MUT-8 and Arg residues of MUT-16 M8BR. Lys residues were observed to be more prone to interact in Martini3. Atomistic simulations revealed that the guanidinium group of Arg also engages in sp2-pi interactions and hydrogen bonds with the backbone of Tyr, making Arg-Tyr interactions stronger than Lys-Tyr, where these additional favorable contacts are absent. In agreement with our simulations, in vitro co-expression pulldown experiments demonstrated a progressive loss of MUT-8 recruitment following the mutation of Arg in MUT-16 M8BR to Lys or Ala, confirming the critical role of Arg in this interaction. These findings advance our understanding of MUT-16 phase separation and subsequent MUT-8 recruitment, key processes in assembling Mutator foci that drive RNA silencing in C. elegans.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
* This version includes new hydration analysis, adding water density maps and radial distribution functions to examine hydration shell thickness in phase-separated condensates. Explicit sodium (Na⁺) and chloride (Cl⁻) molarity quantification was introduced, analyzing their impact on condensate stability and electrostatic screening effects. Additional 350 μs Folding@Home atomistic simulations were incorporated to refine residue-specific interactions, particularly focusing on hydration effects and cation-π interactions. Phase separation assay data were expanded with concentration-dependent observations, specifying MUT-16 FFR phase separation at ≥12.5 μM, M8BR+FFR at ≥6.25 μM, and M8BR alone not phase separating even at 50 μM. Pulldown assays were refined to quantify binding loss in MUT-8 recruitment mutants (R>A and R>K mutations), aligning with computational predictions. Figure 2 was updated for improved visualization of phase separation at different concentrations, and supplementary data were expanded, including additional controls for hydration effects, ion binding, and condensate formation under varying ionic conditions.
* https://zenodo.org/uploads/12742133