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 low-complexity 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 nuclear magnetic resonance spectroscopy. To our surprise, aside from well-studied canonical UBL:UBA interactions, there also exist moderate interactions between the UBL and several disordered regions, including STI1-I and residues 555–570, the latter of which is a known contributor to UBQLN2 LLPS. 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 disordered regions on the phase behavior and physiological functions of UBQLN2.

中文翻译：

---

折叠域和本质上无序域之间以前未表征的相互作用对 UBQLN2 相分离产生不对称影响

穿梭蛋白 UBQLN2 通过其 N 端泛素样 (UBL) 和 C 端泛素相关 (UBA) 结构域分别与蛋白酶体受体和泛素化底物结合，在蛋白质质量控​​制 (PQC) 中发挥作用。在这两个折叠结构域之间是低复杂性的 STI1-I 和 STI1-II 区域，由无序的接头连接。STI1 区域结合对 UBQLN2 的 PQC 功能很重要的其他成分，例如 HSP70。我们最近确定 STI1-II 区域使 UBQLN2 能够进行液-液相分离 (LLPS) 以在体外形成液滴和在细胞中形成生物分子凝聚物。然而，折叠（UBL/UBA）域和本质上无序区域之间的相互作用如何介导相分离在很大程度上是未知的。使用工程域删除结构，我们发现去除 UBA 结构域会抑制 UBQLN2 LLPS，而去除 UBL 结构域会增强 LLPS，这表明 UBA 和 UBL 结构域在调节 UBQLN2 LLPS 中的贡献不对称。为了解释这些差异效应，我们使用核磁共振波谱研究了整个 UBQLN2 分子中涉及 UBA 和 UBL 结构域的相互作用。令我们惊讶的是，除了经过充分研究的规范 UBL：UBA 相互作用外，UBL 与几个无序区域之间也存在适度的相互作用，包括 STI1-I 和残基 555-570，后者是 UBQLN2 LLPS 的已知贡献者。我们的发现对于理解 UBQLN2 LLPS 的分子驱动力和配体与 UBL、UBA、