Unraveling Molecular Interactions in Liquid-Liquid Phase-Separation of Disordered Proteins by Atomistic Simulations.

Membraneless organelles are dynamical cellular condensates formed via biomolecular liquid-liquid phase separation. Despite the blooming research activity in this field, the structural characterization of these entities is very limited and we still do not understand how the phase behaviour is encoded in the amino-acid sequences of the disordered proteins that drive the demixing. Here we exploited explicit-solvent atomistic simulations to investigate the N-terminal disordered region of DEAD-box helicase 4 (NDDX4), which is a well-established model for phase separation. Notably, we determined NDDX4 conformational ensemble at the single-molecule level and we relied on a 'divide-and-conquer' strategy, based on simulations of various protein fragments at high concentration, to probe intermolecular interactions in conditions mimicking real condensates. Our results provide a high-resolution picture of the molecular mechanisms underlying phase separation in agreement with NMR and mutagenesis data and suggest that clusters of arginine and aromatic residues may stabilize the assembly of several condensates.