Ligand Effects on Phase Separation of Multivalent Macromolecules

Abstract Biomolecular condensates enable spatial and temporal control over cellular processes by concentrating biomolecules into non-stoichiometric assemblies. Many condensates form via reversible phase transitions of condensate-specific multivalent macromolecules known as scaffolds. Phase transitions of scaffolds can be regulated by changing the concentrations of ligands, which are defined as non-scaffold molecules that bind to specific sites on scaffolds. Here, we use theory and computation to uncover rules that underlie ligand-mediated control over scaffold phase behavior. We use the stickers -and- spacers model wherein reversible non-covalent crosslinks among stickers drive phase transitions of scaffolds, and spacers modulate the driving forces for phase transitions. We find that the modulatory effects of ligands are governed by: the valence of ligands; whether they bind directly to stickers versus spacers; and the relative affinities of ligand-scaffold versus scaffold-scaffold interactions. In general, all ligands have a diluting effect on the concentration of scaffolds within condensates. Whereas monovalent ligands destabilize condensates, multivalent ligands can stabilize condensates by binding directly to spacers or destabilize condensates by binding directly to stickers. Bipartite ligands that bind to stickers and spacers can alter the structural organization of scaffold molecules within condensates even when they have a null effect on condensate stability. Our work highlights the importance of measuring dilute phase concentrations of scaffolds as a function of ligand concentration in cells. This can reveal whether ligands modulate scaffold phase behavior by enabling or suppressing phase separation at endogeneous levels thereby regulating the formation and dissolution of condensates in vivo . Significance Phase transitions of multivalent macromolecules known as scaffolds help drive the formation of functional biomolecular condensates in cells. The formation and dissolution of condensates is tightly regulated, as aberrant phase behavior is associated with disease. Here, we show that distinct types of ligands can exert control over the formation and dissolution of condensates by binding to distinct sites on scaffold molecules. We further show that the extent and direction of regulation can be inferred through direct measurements of how ligands impact scaffold phase boundaries. Our findings have broad implications for understanding and modeling ligand-mediated regulation of condensates in cells, and for designing novel molecules that exert regulatory control over condensates.