Electrostatics of salt-dependent re-entrant phase behaviors highlights diverse roles of ATP in biomolecular condensates.

Liquid-liquid phase separation (LLPS) involving intrinsically disordered protein regions (IDRs) is a major physical basis for biomolecular condensates such as intracellular membraneless compartments. To elucidate the multifaceted underlying electrostatic effects, which are often essential, salt- and ATP-dependent LLPS of an IDR of the messenger RNA-regulating protein Caprin1 and its phosphorylated variant pY-Caprin1 is investigated. We found that sodium chloride (NaCl) enhances Caprin1 but attenuates pY-Caprin1 LLPS. By comparison, ATP-magnesium (ATP-Mg) also diminishes pY-Caprin1 LLPS monotonically; but [ATP-Mg]-dependent Caprin1 LLPS exhibits reentrance wherein a nonzero [ATP-Mg] is needed for LLPS but Caprin1-rich droplets dissolve at a higher [ATP-Mg]. ATP and Mg ions are much more concentrated in the Caprin1-rich phase than in the dilute phase, whereas Na and Cl ion concentrations are less biased. Similar trends are observed for several other IDRs. Recognizing that Caprin1 has a substantial net positive charge whereas pY-Caprin1 is nearly overall neutral, these experimental trends are rationalized by analytical theory formulated for both polyelectrolytes and polyampholytes, explicit-ion molecular dynamics, and field-theoretic simulations. The models indicate consistently that salt bridges likely contribute to effective stabilizing interactions between charged Caprin1 chains and that the high valence of ATP-Mg is a significant factor for its colocalization with Caprin1. In this light, it is not unreasonable to expect that the high concentrations of ATP structurally driven in large measure by electrostatics into some membraneless compartments may facilitate other intracompartment biochemical functions as well.