Effects of Modulating Multivalent Ligand Binding Accessibility & Affinity on Liquid-Liquid Phase Separation

Elucidating the molecular rules of ligand-induced phase transitions is essential to determining the regulation of the formation and disassembly of biomolecular condensates. Ubiquitin-binding shuttle protein Ubiquilin-2 (UBQLN2) undergoes liquid-liquid phase separation (LLPS) in vitro and colocalizes into stress-induced cytoplasmic puncta in vivosuch as stress granules.[1] A recent study from our lab indicated that ubiquitin (Ub), and compact polyUb chains such as K48- & K11-Ub4 can inhibit UBQLN2 LLPS by binding to phase separation-driving UBQLN2 stickers whereas more extended chains such as K63- & M1-Ub4 can promote UBQLN2 phase separation over a large Ub:UBQLN2 range by acting as a multivalent ligand scaffold.[2] Using a designed tetrameric ubiquitin construct (HOTag6-Ub) that resembles a multi-monoubiquitinated substrate, we further probed how alterations to the stickers and spacers of the HOTag6-Ub ligand can modulate UBQLN2 phase behavior. Using turbidity and microscopy experiments, we obtained phase diagrams for LLPS driven by heterotypic interactions between UBQLN2 and HOTag6-Ub. We discovered that, by changing the length and flexibility of the linker that tethers the Ub units to HOTag6, we can tune UBQLN2 LLPS. Single- and double-point mutations to the Ub hydrophobic binding surface (I44 & V70 "stickers") decreased the binding affinity between Ub and UBQLN2, thereby leading to changes in the shape of HOTag6-Ub/UBQLN2 phase diagrams. Using nuclear magnetic resonance spectroscopy (NMR), microscopy and UV-Vis spectroscopic methods we demonstrated that linker conformation, linker flexibility, and interactions with the Ub binding surface are driving factors of UBQLN2/HOTag6-Ub co-phase separation. Overall, our findings highlight how multivalent ligands can tune phase separation behavior of protein systems. [1] Dao, T. P., Kolaitis, R.-M., Kim, H. J., O'Donovan, K., Martyniak, B., Colicino, E., Hehnly, H., Taylor, J. P., and Castañeda, C. A. (2018). Ubiquitin Modulates Liquid-Liquid Phase Separation of UBQLN2 via Disruption of Multivalent Interactions. Mol. Cell 69, 965-978. [2] Dao, T. P., Yang, Y., Cosgrove, M. S., Hopkins, J. B., Ma, W., Castaneda, C. A. (2021). Mechanistic insights into the enhancement or inhibition of phase separation by polyubiquitin chains of different lengths or linkages. bioRxiv 2021.11.12.467822; doi: https://doi.org/10.1101/2021.11.12.467822. © FASEB.