The structure of the deubiquitinase USP15 reveals a misaligned catalytic triad and an open ubiquitin-binding channel.
The structure of the deubiquitinase USP15 reveals a misaligned catalytic triad and an open ubiquitin-binding channel.
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DOI：
10.1074/jbc.ra118.003857
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发表时间：
2018-11-09
期刊：
影响因子：
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通讯作者：
Dreveny I
中科院分区：
文献类型：
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作者：
Ward SJ;Gratton HE;Indrayudha P;Michavila C;Mukhopadhyay R;Maurer SK;Caulton SG;Emsley J;Dreveny I
泛素特异性蛋白酶15（USP 15）调节重要的细胞过程，包括转化生长因子β（TGF-β）信号传导、线粒体自噬、mRNA加工和先天免疫反应;然而，目前无法获得USP 15催化结构域的结构信息。在这里，我们确定了USP 15催化核心结构域的晶体结构，揭示了一个典型的USP折叠，包括手指，手掌和拇指区域。与parasoline USP 4的结构不同，催化三联体处于无活性构型，催化半胱氨酸与催化组氨酸相距约10个碱基。这种构象是非典型的，并且迄今为止仅在USP 7中观察到类似的未对齐催化三联体，尽管USP 15和USP 7受到不同的调节。此外，我们发现活性位点环是灵活的，导致在很大程度上开放的泛素尾部结合通道。USP 15和USP 4结构的比较指出了可能的活化机制。这两个USP之间的序列差异主要映射到可能赋予特异性的S1′区域，而S1泛素结合口袋高度保守。等温滴定量热法monoubiquitin和线性双泛素结合实验表明，它们的热力学特征显着差异，与USP 15显示出较低的亲和力monoubiquitin比USP 4。此外，我们报告说，USP 15是弱抑制剂米托蒽醌在体外。USP 15-米托蒽醌复合物结构揭示了蒽二酮与S1′结合位点相互作用。我们的研究结果揭示了USP 15的催化结构域结构，构象变化，旁系同源物之间的差异和小分子相互作用的第一个见解，并建立了细胞探针和抑制剂开发的框架。
Ubiquitin-specific protease 15 (USP15) regulates important cellular processes, including transforming growth factor β (TGF-β) signaling, mitophagy, mRNA processing, and innate immune responses; however, structural information on USP15's catalytic domain is currently unavailable. Here, we determined crystal structures of the USP15 catalytic core domain, revealing a canonical USP fold, including a finger, palm, and thumb region. Unlike for the structure of paralog USP4, the catalytic triad is in an inactive configuration with the catalytic cysteine ∼10 Å apart from the catalytic histidine. This conformation is atypical, and a similar misaligned catalytic triad has so far been observed only for USP7, although USP15 and USP7 are differently regulated. Moreover, we found that the active-site loops are flexible, resulting in a largely open ubiquitin tail–binding channel. Comparison of the USP15 and USP4 structures points to a possible activation mechanism. Sequence differences between these two USPs mainly map to the S1′ region likely to confer specificity, whereas the S1 ubiquitin–binding pocket is highly conserved. Isothermal titration calorimetry monoubiquitin- and linear diubiquitin-binding experiments showed significant differences in their thermodynamic profiles, with USP15 displaying a lower affinity for monoubiquitin than USP4. Moreover, we report that USP15 is weakly inhibited by the antineoplastic agent mitoxantrone in vitro. A USP15–mitoxantrone complex structure disclosed that the anthracenedione interacts with the S1′ binding site. Our results reveal first insights into USP15's catalytic domain structure, conformational changes, differences between paralogs, and small-molecule interactions and establish a framework for cellular probe and inhibitor development.