RavN is a member of a previously unrecognized group of Legionella pneumophila E3 ubiquitin ligases.
RavN is a member of a previously unrecognized group of Legionella pneumophila E3 ubiquitin ligases.
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DOI：
10.1371/journal.ppat.1006897
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发表时间：
2018-03
期刊：
影响因子：
6.7
通讯作者：
Machner MP
中科院分区：
文献类型：
--
作者：
Lin YH;Lucas M;Evans TR;Abascal-Palacios G;Doms AG;Beauchene NA;Rojas AL;Hierro A;Machner MP
真核泛素化机制催化小蛋白修饰物泛素与细胞靶蛋白的共价结合，从而改变它们的命运。微生物病原体通过编码E3泛素连接酶的分子模拟来利用这一翻译后修饰过程，E3泛素连接酶是催化泛素化级联反应的最后一步的真核生物酶。在这里，我们证明了嗜肺军团菌效应蛋白Ravn属于一类越来越多的细菌蛋白，它模仿宿主细胞E3连接酶来利用泛素化途径。Ravn的E3连接酶活性位于其N-末端区域，并依赖于与特定的E2泛素结合酶亚群的相互作用。Ravn N-末端区域的晶体结构揭示了一个类似U-box的基序，与其他U-box结构域仅有很小的相似之处，这表明Ravn是一个经历了显著进化变化的E3连接酶残留物。预测的E2结合界面内残基的替换使Ravn处于不活跃状态，这表明尽管结构发生了重大变化，但E2识别模式仍然保守。使用基于隐马尔可夫模型的二级结构分析，我们鉴定并实验验证了另外四个以前没有被认为具有E3连接酶活性的嗜肺乳杆菌效应子，包括SidC的一个新的准对数Lpg2452/SdcB。我们的研究提供了强有力的证据，证明嗜肺乳杆菌正在将相当一部分的效应器武器库用于操纵宿主泛素化途径。细菌病原体经常通过编码真核酶的分子模拟蛋白来劫持保守的宿主途径，从而诱使宿主细胞将其资源交给细菌。在这里，我们展示了细胞内病原体嗜肺军团菌使用这样的策略来利用泛素化，这是一种由E3泛素连接酶介导的保守的翻译后修饰。嗜肺乳杆菌编码宿主E3连接酶的分子模拟物，包括效应蛋白Ravn，从而在感染过程中颠覆泛素化途径。利用蛋白质结晶学，我们发现Ravn的折叠与传统的真核E3只有剩余的相似之处，但它与E2酶的相互作用模式在整个进化过程中都被保留了下来。受Ravn的发现启发，我们进行了电子折叠同源搜索，并在嗜肺性乳杆菌的效应器库中发现了几个额外的E3连接酶候选者，到目前为止，由于缺乏初级序列相似性，这些候选者一直被隐藏着。我们的研究支持这一假设，即E3连接酶是嗜肺乳杆菌毒力计划的重要组成部分，尽管经历了广泛的进化变化，这些效应器仍然保留了对其生物学功能至关重要的特征，包括劫持作为泛素化机制一部分的宿主因子的能力。
The eukaryotic ubiquitylation machinery catalyzes the covalent attachment of the small protein modifier ubiquitin to cellular target proteins in order to alter their fate. Microbial pathogens exploit this post-translational modification process by encoding molecular mimics of E3 ubiquitin ligases, eukaryotic enzymes that catalyze the final step in the ubiquitylation cascade. Here, we show that the Legionella pneumophila effector protein RavN belongs to a growing class of bacterial proteins that mimic host cell E3 ligases to exploit the ubiquitylation pathway. The E3 ligase activity of RavN was located within its N-terminal region and was dependent upon interaction with a defined subset of E2 ubiquitin-conjugating enzymes. The crystal structure of the N-terminal region of RavN revealed a U-box-like motif that was only remotely similar to other U-box domains, indicating that RavN is an E3 ligase relic that has undergone significant evolutionary alteration. Substitution of residues within the predicted E2 binding interface rendered RavN inactive, indicating that, despite significant structural changes, the mode of E2 recognition has remained conserved. Using hidden Markov model-based secondary structure analyses, we identified and experimentally validated four additional L. pneumophila effectors that were not previously recognized to possess E3 ligase activity, including Lpg2452/SdcB, a new paralog of SidC. Our study provides strong evidence that L. pneumophila is dedicating a considerable fraction of its effector arsenal to the manipulation of the host ubiquitylation pathway. Bacterial pathogens often hijack conserved host pathways by encoding proteins that are molecular mimics of eukaryotic enzymes, thus tricking the host cell into surrendering its resources to the bacteria. Here, we show that the intracellular pathogen Legionella pneumophila uses such a strategy to exploit ubiquitylation, a conserved post-translational modification that is mediated by E3 ubiquitin ligases. L. pneumophila encodes molecular mimics of host E3 ligases, including the effector protein RavN, thereby subverting the ubiquitylation pathway for its own benefit during infection. Using protein crystallography, we show that the fold of RavN has only residual resemblance to conventional eukaryotic E3s, yet its mode of interaction with E2 enzymes, host proteins that are important for the ubiquitin transfer reaction, has been preserved throughout evolution. Inspired by the discovery of RavN, we performed an in silico fold homology search and discovered several additional E3 ligase candidates within the effector repertoire of L. pneumophila that, until now, had remained hidden due to lack of primary sequence similarity. Our study supports the hypothesis that E3 ligases are a vital part of the virulence program of L. pneumophila, and that these effectors, despite having undergone extensive evolutionary changes, have retained features that are critical for their biological function, including the ability to hijack host factors that are part of the ubiquitylation machinery.
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影响因子：
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作者：
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影响因子：
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作者：
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Isberg RR
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作者：
Diao, Jianbo;Zhang, Ying;Chen, Jue
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影响因子：
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作者：
Burstein, David;Amaro, Francisco;Zusman, Tal;Lifshitz, Ziv;Cohen, Ofir;Gilbert, Jack A.;Pupko, Tal;Shuman, Howard A.;Segal, Gil
通讯作者：
Segal, Gil
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH