Single Amino Acid Substitutions in Stickers, but Not Spacers, Substantially Alter UBQLN2 Phase Transitions and Dense Phase Material Properties

Abstract

UBQLN2 450−624 oligomerizes and undergoes temperature-responsive liquid−liquid phase transitions following a closed-loop temperature−concentration phase diagram. We recently showed that disease-linked mutations to UBQLN2 450−624 impart highly varying effects to its phase behavior, ranging from little change to significant decrease of saturation concentration and formation of gels and aggregates. However, how single mutations lead to these properties is unknown. Here, we use UBQLN2 450−624 as a model system to study the sequence determinants of phase separation. We hypothesized that UBQLN2 450−624 regions previously identified to promote its oligomerization are the "stickers" that drive interchain interactions and phase separation. We systematically investigated how phase behavior is affected by all 19 possible single amino acid substitutions at three sticker and two "spacer" (sequences separating stickers) positions. Overall, substitutions to stickers, but not spacers, substantially altered the shape of the phase diagram. Within the sticker regions, increasing hydrophobicity decreased saturation concentrations at low temperatures and enhanced oligomerization propensity and viscoelasticity of the dense phase. Conversely, substitutions to acidic residues at all positions greatly increased saturation concentrations. Our data demonstrate that single amino acid substitutions follow a molecular code to tune phase transition behavior of biopolymers.

Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior

“…S1 C and D). To study protein phase behavior, we used a temperature-dependent turbidity assay (18,21,41,42), in which protein solutions are cooled from above to below their phase-transition temperature. Proteins transition from wellmixed to demixed upon cooling below the saturation temperature (T sat ), defined as the point where we first observe an increase in the measured solution turbidity from that of the wellmixed solution.…”

Ligand Effects on Phase Separation of Multivalent Macromolecules

“…Recent studies suggest that scaffolds, which are multivalent macromolecules, can be thought of as biological instantiations of associative polymers (23,24) and modeled using the socalled stickers-and-spacers framework (4, [25][26][27][28][29]. Accordingly, the valence (number) of stickers, the sticker interaction strengths, and the solvation properties of spacers contribute as determinants of the phase behavior of multivalent protein and RNA molecules (30)(31)(32).…”

Key role of UBQLN2 in pathogenesis of amyotrophic lateral sclerosis and frontotemporal dementia

“…Inversely, mutations to hydrophile residues (A488T, P500S, P506S, P509S, P525S) have small impact on oligomerization [10]. Increasing the hydrophobicity within “stickers”, but not “spacers”, favorized the oligomerization tendency of UBQLN2 [67]. The sequence position also greatly impacts UBQLN2 capacity for oligomerization.…”