Previously uncharacterized interactions between the folded and intrinsically disordered domains impart asymmetric effects on UBQLN2 phase separation

Abstract

Shuttle protein UBQLN2 functions in protein quality control (PQC) by binding to proteasomal receptors and ubiquitinated substrates via its N-terminal ubiquitin-like (UBL) and C-terminal ubiquitin-associated (UBA) domains, respectively. Between these two folded domains are intrinsically disordered STI1-I and STI1-II regions, connected by disordered linkers. The STI1 regions bind other components, such as HSP70, that are important to the PQC functions of UBQLN2. We recently determined that the STI1-II region enables UBQLN2 to undergo liquid-liquid phase separation (LLPS) to form liquid droplets in vitro and biomolecular condensates in cells. However, how the interplay between the folded (UBL/UBA) domains and the intrinsically-disordered regions mediates phase separation is largely unknown. Using engineered domain deletion constructs, we found that removing the UBA domain inhibits UBQLN2 LLPS while removing the UBL domain enhances LLPS, suggesting that UBA and UBL domains contribute asymmetrically in modulating UBQLN2 LLPS. To explain these differential effects, we interrogated the interactions that involve the UBA and UBL domains across the entire UBQLN2 molecule using NMR spectroscopy. To our surprise, aside from well-studied canonical UBL:UBA interactions, there also exist moderate and weak interactions between the UBL and STI1-I/STI1-II domains, and between the UBA domain and the linker connecting the two STI1 regions, respectively. Our findings are essential for the understanding of both the molecular driving forces of UBQLN2 LLPS and the effects of ligand binding to UBL, UBA, or STI1 domains on the phase behavior and physiological functions of UBQLN2.

Folded domain charge properties influence the conformational behavior of disordered tails

“…Our results reveal a complex relationship between the effects of FD surface charge distribution, tail length, and tail net charge on IDR-FD interactions. In addition to charge-mediated interactions, this is growing evidence that additional modes of interaction, such as transient hydrophobically-mediated contacts, can also influence how IDRs interact with folded domains ( Sankaranarayanan et al., 2021 ; Cubuk et al., 2021 ; Zheng and Castañeda, 2021 ). In short, the details of FD-IDR interactions are inherently complex and eminently tunable by post-translational modifications or changes in the solution environment ( Martin et al., 2021 ; Moses et al., 2020 ).…”