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.

中文翻译：

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折叠域和固有无序域之间以前未知的相互作用赋予UBQLN2相分离不对称的影响

穿梭蛋白质UBQLN2通过分别通过其N末端泛素样（UBL）和C末端泛素相关（UBA）域与蛋白酶体受体和泛素化底物结合，在蛋白质质量控​​制（PQC）中起作用。在这两个折叠域之间是通过无序接头连接的内在无序的STI1-I和STI1-II区。STI1区域绑定了对UBQLN2的PQC功能很重要的其他组件，例如HSP70。我们最近确定，STI1-II区使UBQLN2能够进行液-液相分离（LLPS），以在体外形成液滴并在细胞中形成生物分子冷凝物。但是，折叠的（UBL / UBA）域和固有无序区域之间的相互作用如何介导相分离是非常未知的。使用工程化的域名删除结构，我们发现，去除UBA域可抑制UBQLN2 LLPS，而去除UBL域可增强LLPS，这表明UBA和UBL域在调节UBQLN2 LLPS中起不对称作用。为了解释这些不同的影响，我们使用NMR光谱法研究了涉及整个UBQLN2分子的UBA和UBL结构域的相互作用。令我们惊讶的是，除了经过深入研究的规范的UBL：UBA相互作用之外，UBL和STI1-I / STI1-II域之间以及UBA域和连接两个STI1区的连接子之间也分别存在中等和弱相互作用。我们的发现对于理解UBQLN2 LLPS的分子驱动力以及配体结合UBL，UBA或STI1域对UBQLN2的相行为和生理功能的影响至关重要。