Sequence Determinants of Intracellular Phase Separation by Complex Coacervation of a Disordered Protein

Abstract

Summary Liquid-liquid phase separation, driven by collective interactions among multivalent and intrinsically disordered proteins, is thought to mediate the formation of membrane-less organelles in cells. Using parallel cellular and in vitro assays we show that the Nephrin intracellular domain (NICD), a disordered protein, drives intracellular phase separation via complex coacervation, whereby the negatively charged NICD co-assembles with positively charged partners to form protein-rich dense liquid droplets. Mutagenesis reveals that the driving force for phase separation depends on the overall amino acid composition and not the precise sequence of NICD. Instead, phase separation is promoted by one or more regions of high negative charge density and aromatic/hydrophobic residues that are distributed across the protein. Many disordered proteins share similar sequence characteristics with NICD, suggesting that complex coacervation may be a widely used mechanism to promote intracellular phase separation.

Direct observation of structure and dynamics during phase separation of an elastomeric protein

“…To probe for intermolecular contacts, 13 C and 12 C edited 1 H-1 H NOESY spectra were recorded for monomeric and coacervated samples containing a 1:1 mixture of unlabeled and 13 C labeled ELP 3 (Fig. 5).…”

“…Chemical shift assignment of the phase-separated state was performed on 7.8 mM ELP 3 with 600 mM NaCl at 37°C. Both the monomer and coacervate 1 H, 13 C, and 15 N chemical shifts were assigned using standard 2D and 3D NMR methods. The 1 H-15 N HSQC, HNCA, HN(CO)CA, HNCO, and HN(CA)CO experiments (pulse sequences as provided by Bruker Biospin) were used for sequential backbone assignments, along with an HCAN experiment (54), which was required to obtain the backbone 15 N chemical shifts of proline residues and to facilitate assignment of proline-rich segments.…”

“…The secondary structure propensity of the major repeating units of the crosslinking and hydrophobic domains in both the monomer and coacervate was predicted using the δ2D structure determination web server for intrinsically disordered proteins (45), with the chemical shifts for 1 HN, 15 N, 13 Cα, 13 (55). Experiments with mixing times of 50, 100, and 150 ms were recorded and used to determine the linear region of the NOE buildup curve.…”

“…Several recent studies have shown that a combination of electrostatic, cation-π, and hydrophobic interactions can drive complex coacervation of intrinsically disordered RNP-associated proteins, highlighting the enthalpic nature of this type of LLPS (13)(14)(15). Conversely, simple coacervation is considered an entropically driven process that depends entirely on hydrophobic interactions between protein chains (4,16), although few molecular details of this process have been reported.…”

Phosphorylation of the FUS low‐complexity domain disrupts phase separation, aggregation, and toxicity

“…However, it is clear that low‐complexity domains specify and mediate functional protein–protein interactions and pathological self‐aggregation. Low‐complexity domains are well‐established targets of post‐translational modifications (Martin et al , 2016), and their sequences specify homotypic and heterotypic interactions mediating liquid–liquid phase separation (LLPS; Lin et al , 2015; Pak et al , 2016). Hence, cells might employ post‐translational modification to “change” the amino acid sequence of prion‐like domains and thus disrupt self‐interaction.…”