Hidden Complexities in the Phase Behavior of Low-Complexity Disordered Proteins

Abstract

Liquid-liquid phase separation (LLPS) is the underlying driving force for membrane-less compartmentalization in cells. LLPS plays key roles in fundamental cell biological processes and is often mediated by disordered low-complexity domains (LCDs). How the driving forces for LLPS are encoded in LCDs and how the phase behavior can be predicted from their sequence is incompletely understood. We are using the stickers-and-spacers framework to reconstruct the full phase behavior. Stickers form non-covalent inter- and intramolecular crosslinks, whereas spacers either enable or suppress the formation of these crosslinks. We have previously shown that aromatic residues are the stickers in the polar prion-like LCD of hnRNPA1. However, the balance of tyrosine and phenylalanine residues varies across LCDs and charged residues may contribute to LLPS in sequences with higher charge content. Herein we (i) extend the stickers-and-spacers model to account for interactions of charged residues and (ii) disentangle the interaction strength of phenylalanine and tyrosine for themselves and for each other. We demonstrate that tyrosine is a stronger sticker than phenylalanine and negatively charged residues are solubilizing spacers. Arginines act as stickers through pairwise interactions with aromatic residues. Importantly, low or high values of the net charge per residue weaken the driving force for LLPS via mean-field electrostatic effects. We further demonstrate that the phase behavior correlates well with the single-chain dimensions of LCD variants with different fractions of aromatic residues, but that this correlation can be broken by mean-field electrostatic effects. Our results support that the stickers-and-spacers model can be adapted for LCDs with a wide range of sequence characteristics, promising precise prediction of phase behavior from the sequence alone.