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

Abstract

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.

A Tug of War Between Condensate Phases in a Minimal Macromolecular System

“…As support for the forgoing classification of regulators, using pentamers of SH3 domains and proline-rich motifs (SH35 and PRM5) as driver proteins 50 , we found Ficoll, lysozyme, and heparin as representatives of volume-exclusion promotors, weak-attraction suppressors, and strong-attraction promotors 51 . This work highlights regulator-driver interaction strength as a key determinant for regulatory effects on thermodynamic 7 properties of phase separation.…”

“…In our previous study 51 , we determined H as a strong-attraction promotor of S:P phase separation. Correspondingly, at S:P equimolar ratio, increasing H initially leads to a decrease in the S and P threshold concentration for phase separation but, upon a further increase in concentration, H switches from a promotor to a suppressor and hence the S and P threshold concentration starts to increase ( Fig.…”

“…Other components presumably play regulatory roles, and still others are recruited as clients but may nevertheless have regulatory effects. For the thermodynamics of liquid-liquid phase separation, computational studies led us to the notion that macromolecular regulators fall into three archetypical classes 15,51 . Volumeexclusion promotors act by taking up space in the bulk phase and thereby displacing driver proteins into the droplet phase.…”

“…This work highlights regulator-driver interaction strength as a key determinant for regulatory effects on thermodynamic 7 properties of phase separation. Macromolecular components can also affect the material properties and time evolution of condensates 19,32,37,42,[47][48][49]51 {Maharana, 2018 #75}{Mann, 2019 #21}, but the underlying physical rules are far from clear. Here we uncover some of these rules by studying the phase behaviors of a macromolecular system comprising just four components, SH35, PRM5, lysozyme, and heparin.…”

Prediction of liquid-liquid phase separation proteins using machine learning

“…More and more studies suggested that many cellular metabolic processes are regulated by LLPS, so are some intractable diseases such as ALS (amyotrophic lateral sclerosis) [4] and AD (Alzheimer disease) [5]. A number of proteins have been verified to be able to form liquid-like membraneless assemblies in cells and in vitro [3,[6][7][8][9]. Meanwhile, studies indicated that RNA can regulate the LLPS of proteins and influence the formation of bio-molecular condensates [10].…”