Comparative Roles of Charge, $π$ and Hydrophobic Interactions in Sequence-Dependent Phase Separation of Intrinsically Disordered Proteins (2005.06712v2)

Abstract: Endeavoring toward a transferable, predictive coarse-grained explicit-chain model for biomolecular condensates underlain by liquid-liquid phase separation (LLPS), we conducted multiple-chain simulations of the N-terminal intrinsically disordered region (IDR) of DEAD-box helicase Ddx4, as a test case, to assess the roles of electrostatic, hydrophobic, cation-$\pi$, and aromatic interactions in amino acid sequence-dependent LLPS. We evaluated 3 residue-residue interaction schemes with a shared electrostatic potential. Neither a common hydrophobicity scheme nor one augmented with arginine/lysine-aromatic cation-$\pi$ interactions consistently accounted for the experimental LLPS data on the wildtype, a charge-scrambled, an FtoA, and an RtoK mutant of Ddx4 IDR. In contrast, interactions based on contact statistics among folded globular protein structures reproduce the overall experimental trend, including that the RtoK mutant has a much diminished LLPS propensity. Consistency between simulation and LLPS experiment was also found for RtoK mutants of P-granule protein LAF-1, underscoring that, to a degree, the important LLPS-driving $\pi$-related interactions are embodied in classical statistical potentials. Further elucidation will be necessary, however, especially of phenylalanine's role in condensate assembly because experiments on FtoA and YtoF mutants suggest that LLPS-driving phenylalanine interactions are significantly weaker than those posited by common statistical potentials. Protein-protein electrostatic interactions are modulated by relative permittivity, which depends on protein concentration. Analytical theory suggests that this dependence entails enhanced inter-protein interactions in the condensed phase but more favorable protein-solvent interactions in the dilute phase. The opposing trends lead to a modest overall impact on LLPS.

Comparative Roles of Charge, π, and Hydrophobic Interactions in Sequence-Dependent Phase Separation of Intrinsically Disordered Proteins

The paper presents a detailed computational paper on the liquid-liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs), focusing on the N-terminal intrinsically disordered region (IDR) of the DEAD-box helicase Ddx4 as a model system. The research examines the roles of electrostatic, hydrophobic, cation-π, and aromatic interactions in driving LLPS, considering both wildtype and specific mutant sequences of Ddx4 IDR, including charge-scrambled, phenylalanine-to-alanine, and arginine-to-lysine variants.

The paper employs coarse-grained, residue-based models to simulate the phase behavior of these IDRs and evaluates several theoretical interaction schemes, including those based on hydrophobicity, cation-π terms augmented models, and structure-derived statistical potentials. Notably, it finds that the latter, particularly the Kim-Hummer (KH) model based on statistical potentials from globular protein structures, offers the best agreement with experimental LLPS data, capturing key effects such as the reduced phase separation propensity observed in R-to-K mutants.

The simulations reveal that while hydrophobic and electrostatic interactions contribute to LLPS, they are insufficient on their own to explain observed experimental trends. Instead, the findings underscore the critical role of π-related interactions, particularly those involving arginine over lysine. This preference is partly rationalized by the geometrical capability of arginine to form more favorable contact configurations.

A significant observation is that phenylalanine-related interactions, predicted to be strongly favorable in folded protein cores, do not translate as strongly to LLPS contexts where residues tend to be more solvent-exposed. This suggests that current statistical potentials may overestimate the interaction strength of phenylalanine in phase-separated environments, pointing to the need for refined models that account for the unique contact environment in LLPS.

Furthermore, the paper explores the concept of concentration-dependent permittivity within phase-separated droplets, suggesting it can modulate protein self-interactions modestly, despite having potentially significant functional implications. This highlights the complexity of electrostatic influences in biologically relevant LLPS scenarios and suggests future research could better delineate these effects.

In conclusion, the paper advances our understanding of LLPS by demonstrating that a combined electrostatic, π-related interaction perspective, informed by empirical statistical potentials from folded proteins, aligns more closely with experimental observations than models emphasizing solely on hydrophobicity or isolated cation-π interactions. Future research should aim at refining interaction models to better describe solvent-exposed hydrophobic/aromatic interactions and incorporate dynamic response properties such as temperature effects and ionic contributions. These would further enhance our conceptual and predictive capabilities for biomolecular LLPS, impacting fields from cellular biology to materials science.