Previously uncharacterized interactions between the folded and intrinsically disordered domains impart asymmetric effects on UBQLN2 phase separation
Previously uncharacterized interactions between the folded and intrinsically disordered domains impart asymmetric effects on UBQLN2 phase separation
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DOI：
10.1002/pro.4128
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发表时间：
2021-02
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影响因子：
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通讯作者：
Tongyin Zheng;C. Castañeda
中科院分区：
文献类型：
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作者：
Tongyin Zheng;C. Castañeda
穿梭蛋白UBQLN2通过其N端泛素样结构域(UBL)和C端泛素相关结构域(UBA)分别与蛋白酶体受体和泛素化底物结合，从而在蛋白质质量控制(PQC)中发挥作用。在这两个折叠结构域之间是本质上无序的STI1-I和STI1-II区域，通过无序连接子连接。STI1区域与其他组件结合，如HSP70，这些组件对UBQLN2的PQC功能很重要。我们最近确定，STI1-II区域使UBQLN2能够经历液-液相分离(LLP)，在体外形成液滴，在细胞中形成生物分子凝聚体。然而，折叠(UBL/UBA)结构域和固有无序区之间的相互作用如何调节相分离在很大程度上是未知的。使用工程结构域删除构建，我们发现去除UBA结构域抑制UBQLN2LLP，而去除UBL域增强LLP，这表明UBA和UBL域在调节UBQLN2LLP方面起着不对称的作用。为了解释这些不同的影响，我们使用核磁共振光谱询问了整个UBQLN2分子中涉及UBA和UBL域的相互作用。令我们惊讶的是，除了已经研究得很好的典型Ubl：Uba相互作用外，Ubl和STI1-I/STI1-II结构域之间，以及UBA结构域和连接两个STI1区域的连接子之间也分别存在中等和微弱的相互作用。我们的发现对于理解UBQLN2LLP的分子驱动力以及与UBL、UBA或STI1结构域的配体结合对UBQLN2的相行为和生理功能的影响都是至关重要的。工作声明的影响郑Castañeda表明，折叠结构域和内在无序区域之间的相互作用调节UBQLN2的液-液相分离行为，UBQLN2是一种蛋白质质量控制(PQC)穿梭蛋白。尽管折叠的UBL和UBA结构域大小相似，但由于它们与中间固有无序区的不对称相互作用，它们分别抑制和促进了相分离。这些结果有力地表明，包括蛋白酶体受体在内的PQC成分可能调节细胞中的UBQLN2相分离行为。
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. Impact of Work Statement Zheng and Castañeda show that interplay between the folded domains and intrinsically disordered regions regulates liquid-liquid phase separation behavior of UBQLN2, a protein quality control (PQC) shuttle protein. Despite their similar size, the folded UBL and UBA domains inhibit and promote phase separation, respectively, due to their previously uncharacterized, asymmetric interactions with the middle intrinsically-disordered region. These results strongly suggest that PQC components, including proteasomal receptors, are likely to modulate UBQLN2 phase separation behavior in cells.