Three archetypical classes of macromolecular regulators of protein liquid-liquid phase separation

Membraneless organelles, corresponding to the droplet phase upon liquid-liquid phase separation (LLPS) of protein or protein-RNA mixtures, mediate myriad cellular functions. Cells use a variety of biochemical signals such as expression level and posttranslational modification to regulate droplet formation and dissolution, but the physical basis of the regulatory mechanisms remains ill-defined and quantitative assessment of the effects is largely lacking. Our computational study predicted that the strength of attraction by droplet-forming proteins dictates whether and how macromolecular regulators promote or suppress LLPS. We experimentally tested this prediction, using the pentamers of SH3 domains and proline-rich motifs (SH3₅ and PRM₅) as droplet-forming proteins. Determination of the changes in phase boundary and the partition coefficients in the droplet phase over a wide range of regulator concentrations yielded both a quantitative measure and a mechanistic understanding of the regulatory effects. Three archetypical classes of regulatory effects were observed. Ficoll 70 at high concentrations indirectly promoted SH3₅-PRM₅ LLPS, by taking up volume in the bulk phase and thereby displacing SH3₅ and PRM₅ into the droplet phase. Lysozyme had a moderate partition coefficient and suppressed LLPS by substituting weaker attraction with SH3₅ for the stronger SH3₅-PRM₅ attraction in the droplet phase. By forming even stronger attraction with PRM₅, heparin at low concentrations partitioned heavily into the droplet phase and promoted LLPS. These characteristics were recapitulated by computational results of patchy particle models, validating the identification of the 3 classes of macromolecular regulators as volume-exclusion promotors, weak-attraction suppressors, and strong-attraction promotors.