Solution Structure of the Ubiquitin-associated (UBA) Domain of Human Autophagy Receptor NBR1 and Its Interaction with Ubiquitin and Polyubiquitin
Solution Structure of the Ubiquitin-associated (UBA) Domain of Human Autophagy Receptor NBR1 and Its Interaction with Ubiquitin and Polyubiquitin
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DOI：
10.1074/jbc.m114.555441
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发表时间：
2014-04
期刊：
影响因子：
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通讯作者：
E. Walinda;D. Morimoto;K. Sugase;T. Konuma;H. Tochio;M. Shirakawa
中科院分区：
文献类型：
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作者：
E. Walinda;D. Morimoto;K. Sugase;T. Konuma;H. Tochio;M. Shirakawa
背景：自噬受体NBR1常见于神经退行性疾病泛素阳性包涵体中。结果：描述了NBR1对泛素和多泛素的分子识别作用。结论：NBR1的泛素相关结构域与单泛素有很高的亲和力，但缺乏多泛素的连接特异性。意义：NBR1可能通过与泛素的非连接特异性结合而高效地与泛素化蛋白形成细胞内包涵体。NBR1(BRCA1基因1的邻居)是一种常见于神经退行性疾病泛素阳性包涵体中的蛋白质。由于其结构与研究较多的自噬受体蛋白p62/SQSTM1高度相似，NBR1被认为类似于通过其C端泛素相关(UBA)结构域与泛素标记的自噬底物结合，并将它们运送到自噬小体进行降解。出乎意料的是，我们发现NBR1在UBA结构上与p62不同，因此在与泛素的相互作用方面也不同。在螺旋α-3上观察到结构上的差异，它与螺旋α-2倾斜得更远，并在NBR1中延伸了大约一圈。这不仅抑制了NBR1UBA的p62型自我二聚，而且与p62UBA相比，对Monoubiquitin的亲和力显着提高。重要的是，NBR1UBA-泛素复合体的结构表明，UBA中保守的MGF基序前面的侧链的负电荷在泛素的识别中起着不可或缺的作用。此外，核磁共振和等温滴定量热法实验表明，NBR1UBA以相似的亲和力结合到聚泛素的每个单体单位上，并且通过与单泛素结合的相同表面。这表明NBR1缺乏多泛素连接类型的特异性，这与细胞内泛素阳性包涵体中观察到的非特异性连接很好地一致。因此，我们的结果表明，NBR1 UBA和p62 UBA之间的结构差异导致NBR1对泛素的亲和力要高得多，这反过来表明NBR1可能比p62更有效地与泛素化的自噬底物形成细胞内包涵体。
Background: The autophagic receptor NBR1 is commonly found in ubiquitin-positive inclusions in neurodegenerative diseases. Results: Molecular recognition of ubiquitin and polyubiquitin by NBR1 is described. Conclusion: The ubiquitin-associated domain of NBR1 shows unexpectedly high affinity for monoubiquitin but lacks polyubiquitin linkage specificity. Significance: NBR1 may be highly efficient at forming intracellular inclusions with ubiquitylated proteins via non-linkage-specific association with ubiquitin. NBR1 (neighbor of BRCA1 gene 1) is a protein commonly found in ubiquitin-positive inclusions in neurodegenerative diseases. Due to its high architectural similarity to the well studied autophagy receptor protein p62/SQSTM1, NBR1 has been thought to analogously bind to ubiquitin-marked autophagic substrates via its C-terminal ubiquitin-associated (UBA) domain and deliver them to autophagosomes for degradation. Unexpectedly, we find that NBR1 differs from p62 in its UBA structure and accordingly in its interaction with ubiquitin. Structural differences are observed on helix α-3, which is tilted farther from helix α-2 and extended by approximately one turn in NBR1. This results not only in inhibition of a p62-type self-dimerization of NBR1 UBA but also in a significantly higher affinity for monoubiquitin as compared with p62 UBA. Importantly, the NBR1 UBA-ubiquitin complex structure shows that the negative charge of the side chain in front of the conserved MGF motif in the UBA plays an integral role in the recognition of ubiquitin. In addition, NMR and isothermal titration calorimetry experiments show that NBR1 UBA binds to each monomeric unit of polyubiquitin with similar affinity and by the same surface used for binding to monoubiquitin. This indicates that NBR1 lacks polyubiquitin linkage-type specificity, in good agreement with the nonspecific linkages observed in intracellular ubiquitin-positive inclusions. Consequently, our results demonstrate that the structural differences between NBR1 UBA and p62 UBA result in a much higher affinity of NBR1 for ubiquitin, which in turn suggests that NBR1 may form intracellular inclusions with ubiquitylated autophagic substrates more efficiently than p62.