Determining the molecular mechanism of NDDX4 liquid-liquid phase separation under physiological conditions

• Alejandro Salas-Estrada, Physics, University of Delaware

• Juan Perilla, Chemistry and Biochemistry, University of Delaware

Membrane-less organelles (MLOs) are biomolecular condensates that lack a surrounding lipid membrane and can demonstrate liquid-like properties. They play a crucial role in the cellular landscape by facilitating compartmentalization and regulating essential processes such as cellular and nuclear transport and protein synthesis. Nevertheless, the biomolecular principles underpinning MLO assembly and function remain unclear. Recently, Liquid-Liquid Phase Separation (LLPS) has been recognized as a driver of formation of these structures. Disordered proteins play a determinant role in LLPS because of their multivalent intermolecular interactions and tendency to remain unfolded. Furthermore, single-chain properties of disordered proteins are correlated with their ability to induce phase separation in biological condensates. The present work focuses on the N-terminal of DEAD-box helicase 4 (NDDX4), a disordered scaffold protein extensively studied in the context of phase separation. More specifically, all-atom molecular dynamics simulations of NDDX4 were employed to determine inter-residue dynamics. Our results were validated against previously derived NMR observables (NOE distance restraints) and empirical phase diagrams that probed the interaction network of condensed NDDX4. We found that the conformational ensemble of NDDX4 favors inter-residue contacts between oppositely charged residues and between sequence regions rich in aromatic aminoacids. Our results showcase the power of employing atomistic simulations to establish the role of aromatic, acidic and basic aminoacids in driving LLPS under physiological conditions.