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

Abstract

Membraneless organelles are dynamical cellular condensates formed via biomolecular liquid–liquid phase separation of proteins and RNA molecules. Multiple evidence suggests that in several cases disordered proteins are structural scaffolds that drive the condensation by forming a dynamic network of inter- and intramolecular contacts. 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 behavior is encoded in the amino acid sequences of the scaffolding proteins. 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.

Liquid–Liquid Phase Separation by Intrinsically Disordered Protein Regions of Viruses: Roles in Viral Life Cycle and Control of Virus–Host Interactions

“…Overall, condensate immiscibility appears as a very general feature in biological systems, which could also be exploited to create synthetic membrane-less compartments [127,128]. Theoretical and computational studies on protein de-mixing [127,[129][130][131] confirm that protein phase diagrams are mainly shaped by salt concentration and solvent-mediated interactions, in turn influenced by temperature, their effects being cumulatively translated into "interfacial tension", i.e., the work required to increase the size of the interface between two adjacent, immiscible phases. [127].…”

“…[127]. Sequence-specific properties, such as the presence of charged and aromatic residues, may then stabilize the de-mixing arrangements [130]. The role of salts on protein de-mixing has been considered for the peculiar case of multivalent ions [132,133].…”

Molecular determinants of phase separation forDrosophilaDNA replication licensing factors

“…In addition to overall net charge, recent theoretical and experimental work has demonstrated that the patterning of charged residues within disordered domains can fine-tune phase separation propensity (Lin et al, 2018;Nott et al, 2015;Pak et al, 2016;Paloni et al, 2020). We assessed the distribution of charged residues within the Cdt1 IDR by calculating the net charge per residue (NCPR) over a 5-residue window (Figure 2E, top).…”

Hydrophobic residues advance the onset of simple coacervation in intrinsically disordered proteins at low densities: Insights from field theoretical simulations studies

“…Given the importance of chemical specificity of the sequence and also the importance of solvation, computer simulations with detailed atomic models with explicit solvent would be ideal to test and/or predict LLPS propensities of a given solution. However, the length and time scales are formidable and computationally intractable even for a simple model systems [33]. Coarse-grained (CG) models, with reduced representations of amino acids and solvents, provide a computationally tractable alternative.…”