How do intrinsically disordered protein regions encode a driving force for liquid-liquid phase separation?

Abstract

Liquid-liquid phase separation is the mechanism underlying the formation of biomolecular condensates. Disordered protein regions often drive phase separation, but the molecular interactions mediating this phenomenon are not well understood, sometimes leading to the conflation that all disordered protein regions drive phase separation. Given the critical role of phase separation in many cellular processes, and that dysfunction of phase separation can lead to debilitating diseases, it is important that we understand the interactions and sequence properties underlying phase behavior. A conceptual framework that divides IDRs into interacting and solvating regions has proven particularly useful, and analytical instantiations and coarse-grained models can test our understanding of the driving forces against experimental phase behavior. Validated simulation paradigms enable the exploration of sequence space to help our understanding of how disordered protein regions can encode phase behavior, which IDRs may mediate phase separation in cells, and which IDRs are highly soluble.

Figure 1.. Conceptualizing liquid-liquid phase separation of IDRs.

The interactions that drive LLPS in domain-motif systems and IDRs can both be described by the stickers-and-spacers framework. Stickers are adhesive elements that contribute to the main interaction potential, and they are connected by largely non-interacting spacers. (A) Heterotypic LLPS in domain-motif systems, e.g., between a folded SH3 domain and a proline-rich motif (PRM) (top, PDB ID: 1SEM). LLPS of IDRs can be mediated by a multitude of multivalent interactions. These may include interactions of individual residues or longer motifs, e.g., LARKS (bottom, PDB ID: 6CF4). (B) SH3 tandem repeats connected by linker regions can phase separate in the presence of tandem repeats of PRMs (top). The homotypic intermolecular interactions that drive phase separation of IDRs are satisfied intramolecularly in the dilute phase (bottom). (C) In the stickers-and-spacers framework, SH3 domains and PRMs are stickers, and the connecting linkers are spacers. For IDRs, the single residues or motifs are the stickers and the intervening residues spacers.

Figure 2.. Sticker valence, interaction strength and patterning, and spacer properties determine biomolecule phase behavior.

(A) The schematic phase diagram shows the coexistence curve for a biomolecule as a function of interaction parameter χ, which can, e.g., be modulated by temperature, pH and salt concentration. As the coexistence curve is crossed and the biomolecule enters the two-phase regime, the biomolecule undergoes LLPS and forms a dilute and a dense phase, whose concentrations are given by the left and right arms of the coexistence curve, i.e., c_sat and c_dense. The zenith of the curve is the critical point beyond which no phase separation occurs. Increasing the driving forces for phase separation will result in a widening of the two-phase regime and an increase in the position of the critical point. (B) The sticker valence determines the driving force for LLPS. Increasing the sticker valence increases the driving force for phase separation; too few stickers results in the absence of a two-phase regime. (C) Increasing the sticker interaction strength increases the driving force for LLPS. (D) Increasing the number of spacers between the stickers decreases the driving force for phase separation by reducing the cooperativity of sticker-sticker interactions. (E) The effective solvation volume of spacers determines whether the formation of three-dimensional networks is coupled to a density transition, i.e., LLPS. In the case of well-solvated spacers, stickers can mediate the formation of a system-spanning network even in the absence of LLPS. (F) Clustering of stickers alters multiple properties of the biomolecule simultaneously. It decreases the effective valence of the system, increases the effective interaction strength of the stickers and increases the spacing between the stickers. In the case of aromatic stickers, clustering can promote amorphous aggregation over LLPS.