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

Abstract

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.