Multiscale simulations of molecular recognition by phase separated MUT-16: A scaffolding protein of Mutator foci

Biomolecular recruitment by phase separated condensates has emerged as a key organising principle of biological processes. One such process is the RNA silencing pathway, which regulates gene expression and genomic defense against foreign nucleic acids. In C. elegans , this pathway involves siRNA amplification at perinuclear germ granules named Mutator foci . The formation of Mutator foci depends on the phase separation of MUT-16, acting as a scaffolding protein to recruit other components of the Mutator complex. Earlier studies have indicated a crucial role for an exoribonuclease, MUT-7, in RNA silencing. The recruitment of MUT-7 to Mutator foci is facilitated by a bridging protein, MUT-8. However, how MUT-8 binds to MUT-16 remains elusive. We resolved the molecular drivers of MUT-16 phase separation and the recruitment of MUT-8 using multi-scale molecular dynamics simulations and in vitro experiments. Residue-level coarse-grained simulations predicted the relative phase separation propensities of MUT-16 disordered regions, which we validated by experiments.

Coarse-grained simulations at residue-level and near atomic-resolution also indicated the essential role of aromatic amino acids (Tyr and Phe) in MUT-16 phase separation. Furthermore, coarse-grained and atomistic simulations of MUT-8 N-terminal prion-like domain with phase separated MUT-16 condensate revealed the importance of cation- π interaction between Tyr residues of MUT-8 and Arg/Lys residues of MUT-16. By re-introducing atomistic detail to condensates from coarse-grained and 350 µs all-atom simulations in explicit solvent on Folding@Home, we demonstrate Arg-Tyr interaction surpasses the strength of Lys-Tyr interactions in the recruitment of MUT-8. The atomistic simulations show that the planar guanidinium group of Arg also engages in sp ² - π interaction, and hydrogen bonds with the Tyr residues and these additional favorable contacts are missing in the Lys-Tyr interactions. In agreement with simulations, the mutation of seven Arg residues in MUT-16 to Lys and Ala weakens MUT-8 binding in vitro .