Multi-scale Simulations Reveal Molecular Drivers in MUT-16 Scaffold Protein Phase Separations and Client Recognition

Abstract

AbstractRecruitment of biomolecules by phase-separated condensates has emerged as a fundamental organising principle of biological processes. One such process is the RNA silencing pathway, which regulates gene expression and genomic defence against foreign nucleic acids. InC. elegans, this pathway involves siRNA amplification at perinuclear germ granules namedMutator foci. The formation ofMutator focidepends 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 toMutator fociis 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 andin vitroexperiments. Residue-level coarse-grained simulations predicted the relative phase separation propensities of MUT-16 disordered regions, which we validated by experiments. Near-atomic coarse-grained simulations also capture the relative tendencies of different sequences to phase-separate. Together, coarse-grained simulations at the residue level and near-atomic resolution 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 revealed the importance of cation-πinteraction between Tyr residues of MUT-8 and Arg/Lys residues of MUT-16. By re-introducing atomistic detail into condensates derived from coarse-grained models and conducting 350 µs all-atom simulations 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 sp2-πinteraction, and hydrogen bonds with the Tyr residues and these additional favourable 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 bindingin vitro.