Complex coacervation contributes to the joint phase behaviors of mixtures of charge-deficient prion-like low-complexity domains.

Abstract

Prion-like low-complexity domains (PLCDs) are involved in the formation and regulation of various cellular biomolecular condensates, including stress granules and transcriptional condensates, which form via phase separation coupled to percolation. Our recent work has uncovered how evolutionarily conserved sequence features drive PLCD phase behavior through homotypic interactions. Since many condensates encompass a diverse mixture of proteins with PLCDs, we asked if mixtures of PLCDs have emergent phase behaviors that go beyond the intrinsic phase behaviors that have been well-documented through recent, in-depth biophysical studies. Specifically, we combined coarse-grained, single-bead-per-residue stickers-and-spacers simulations with experimental phase separation assays to study mixtures of PLCDs from the proteins hnRNPA1 and FUS. We refer to these domains as A1-LCD and FUS-LCD, respectively. Surprisingly, we find that a 1:1 mixture of the two species phase separates more readily than either sequence on its own. We demonstrate that the enhanced driving forces for phase separation of mixtures of A1-LCD and FUS-LCD comes from complementary electrostatic interactions between the two proteins. This complex coacervation-like mechanism adds to complementary interactions among aromatic sticker residues. Our findings are striking because we find evidence for an enhancement mediated by complex coacervation even though only 12% and 2.8% of the residues in the A1- and the FUS-LCD are charged. Importantly, the condensates formed by mixtures of A1- and FUS-LCD show inhomogeneous spatial organization whereby molecules are spatially proximal to species with which they have the strongest interactions. This deviates from a random mixture model and instead reflects a fine interplay between homotypic and heterotypic associations. Our findings support the emerging view that multicomponent condensates involve complex networks of homotypic and heterotypic interactions that drive a coupling between associative and segregative phase transitions.