Decoding optimal ligand design for multicomponent condensates

Abstract

Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or post-translational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules for various cellular processes. Specific interactions between polyubiquitin chains of different linkages and partner proteins, including hHR23B, NEMO, and UBQLN2, regulate condensate assembly or disassembly. Here, we used a library of designed polyubiquitin hubs and UBQLN2 as model systems for determining the driving forces of ligand-mediated phase transitions. Systematic decreases to the binding affinity between Ub and UBQLN2 or deviations from the optimal spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. Using an analytical model that accurately described the effects of different hubs on UBQLN2 phase diagrams, we determined that introduction of Ub to UBQLN2 condensates incurs a significant inclusion energetic penalty. This penalty competes with the hub's ability to scaffold multiple UBQLN2 molecules, thereby cooperatively amplifying phase separation. Importantly, there exists an optimal polyubiquitin hub design that maximally promotes phase separation. Hubs where Ub units are too close or too far apart inhibit phase separation. These effects on phase diagrams are encoded in the spacings between Ub units as found for naturally-occurring chains of different linkages and designed chains of different architectures, thus illustrating how the ubiquitin code regulates functionality via the emergent properties of the condensate. We expect our findings to extend to other condensates necessitating the consideration of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates.

Reduction of oligomer size modulates the competition between cluster formation and phase separation of the tumor suppressor SPOP

“…Briefly, some degree of self-association is necessary to amplify weak DAXX-DAXX interactions, but excessive self-association inhibits these same DAXX-DAXX interactions. Recent work on the mechanism of phase separation of Ubiquilin2 (UBQLN2) with poly-ubiquitin chains revealed the same physical principle (45). The soluble polyubiquitin "hub" binds and bundles UBQLN2, which has an intrinsic driving force for phase separation, thereby enhancing phase separation.…”

Reduction of oligomer size modulates the competition between cluster formation and phase separation of the tumor suppressor SPOP

“…Briefly, some degree of self-association is necessary to amplify weak DAXX-DAXX interactions, but excessive self-association inhibits these same DAXX-DAXX interactions. Recent work on the mechanism of phase separation of ubiquilin2 (UBQLN2) with polyubiquitin chains revealed the same physical principle ( 45 ). The soluble polyubiquitin “hub” binds and bundles UBQLN2, which has an intrinsic driving force for phase separation, thereby enhancing phase separation.…”