Structure of the Legionella Virulence Factor, SidC Reveals a Unique PI(4)P-Specific Binding Domain Essential for Its Targeting to the Bacterial Phagosome.
Structure of the Legionella Virulence Factor, SidC Reveals a Unique PI(4)P-Specific Binding Domain Essential for Its Targeting to the Bacterial Phagosome.
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DOI：
10.1371/journal.ppat.1004965
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发表时间：
2015-06
期刊：
影响因子：
6.7
通讯作者：
Mao Y
中科院分区：
文献类型：
--
作者：
Luo X;Wasilko DJ;Liu Y;Sun J;Wu X;Luo ZQ;Mao Y
条件性细胞内病原体嗜肺军团菌是军团病的病原体。L.嗜肺军团菌将近300种效应蛋白递送到宿主细胞中，用于建立被称为含军团菌空泡（LCV）的允许复制的区室。SidC和它的副受体SdcA是两种效应物，已经显示它们通过结合磷脂酰肌醇-4-磷酸[PI（4）P]而锚在LCV上，以促进ER蛋白向LCV的募集。我们最近报道了SidC的N-末端SNL（SidC N-末端E3连接酶）结构域是泛素E3连接酶，并且其活性是ER蛋白向LCV募集所必需的。本文报道了SidC（1-871）的晶体结构。该结构揭示了SidC包含四个结构域，它们被包装成拱形形状。P4 C结构域（SidC的PI（4）P结合）包含四个α-螺旋束并且覆盖SNL结构域的泛素连接酶催化位点。引人注目的是，在该束的一端形成具有特征正静电势的口袋。P4 C结构域的脂质体结合测定进一步鉴定了磷酸肌醇识别和膜相互作用的决定因素。有趣的是，我们还发现与PI（4）P结合刺激E3连接酶活性，推测是由于PI（4）P诱导的构象转换从封闭形式到开放活性形式。参与PI（4）P结合的关键残基的突变显著降低了SidC与LCV的结合，并消除了其在ER蛋白和泛素信号的募集中的活性，突出了PI（4）P介导的SidC靶向对其在细菌吞噬体膜重塑中的功能至关重要。最后，证明了与P4 C结构域的GFP融合物特异性地定位于哺乳动物细胞中富含PI（4）P的隔室。该结构域显示了在活细胞中发展成灵敏和准确的PI（4）P探针的潜力。军团病是由细胞内细菌病原体嗜肺军团菌引起的。这种细菌的成功感染需要一个特殊的分泌系统，将近300种效应蛋白注入宿主细胞的细胞质中。效应子SidC和它的辅基SdcA锚在含军团菌的空泡（LCV）上，并且对于ER蛋白向LCV的募集是重要的。最近的数据表明，SidC和SdcA是泛素E3连接酶，它们的活性是LCV上ER蛋白和泛素缀合物富集所必需的。在这里，我们提出了SidC的晶体结构，揭示了一个新的PI（4）P-结合模块的架构。我们的生物化学和细胞生物学研究突出了PI（4）P-结合和膜插入的关键决定因素。这种新型PI（4）P结合模块的表征为开发精确的体内PI（4）P探针开辟了潜在途径。我们的数据还揭示了一个独特的调节机制的泛素E3连接酶活性的SidC，这是激活的脂质分子，PI（4）P。此外，我们的研究结果表明，适当的空间定位的SidC的细胞质表面的细菌吞噬体通过与PI（4）P的结合是至关重要的，它的功能。
The opportunistic intracellular pathogen Legionella pneumophila is the causative agent of Legionnaires’ disease. L. pneumophila delivers nearly 300 effector proteins into host cells for the establishment of a replication-permissive compartment known as the Legionella-containing vacuole (LCV). SidC and its paralog SdcA are two effectors that have been shown to anchor on the LCV via binding to phosphatidylinositol-4-phosphate [PI(4)P] to facilitate the recruitment of ER proteins to the LCV. We recently reported that the N-terminal SNL (SidC N-terminal E3 Ligase) domain of SidC is a ubiquitin E3 ligase, and its activity is required for the recruitment of ER proteins to the LCV. Here we report the crystal structure of SidC (1-871). The structure reveals that SidC contains four domains that are packed into an arch-like shape. The P4C domain (PI(4)P binding of SidC) comprises a four α-helix bundle and covers the ubiquitin ligase catalytic site of the SNL domain. Strikingly, a pocket with characteristic positive electrostatic potentials is formed at one end of this bundle. Liposome binding assays of the P4C domain further identified the determinants of phosphoinositide recognition and membrane interaction. Interestingly, we also found that binding with PI(4)P stimulates the E3 ligase activity, presumably due to a conformational switch induced by PI(4)P from a closed form to an open active form. Mutations of key residues involved in PI(4)P binding significantly reduced the association of SidC with the LCV and abolished its activity in the recruitment of ER proteins and ubiquitin signals, highlighting that PI(4)P-mediated targeting of SidC is critical to its function in the remodeling of the bacterial phagosome membrane. Finally, a GFP-fusion with the P4C domain was demonstrated to be specifically localized to PI(4)P-enriched compartments in mammalian cells. This domain shows the potential to be developed into a sensitive and accurate PI(4)P probe in living cells. Legionnaires’ disease is caused by the intracellular bacterial pathogen Legionella pneumophila. Successful infection by this bacterium requires a special secretion system that injects nearly 300 effector proteins into the cytoplasm of host cells. The effector SidC and its paralog SdcA anchor on the Legionella-containing vacuole (LCV) and are important for the recruitment of ER proteins to the LCV. Recent data demonstrated that SidC and SdcA are ubiquitin E3 ligases and that their activity is required for the enrichment of ER proteins and ubiquitin conjugates on the LCV. Here we present the crystal structure of SidC revealing the architecture of a novel PI(4)P-binding module. Our biochemical and cell biological studies highlight key determinants involved in PI(4)P-binding and membrane insertion. Characterization of this novel PI(4)P binding module opens a potential avenue for the development of an accurate in vivo PI(4)P probe. Our data also reveals a distinct regulatory mechanism of the ubiquitin E3 ligase activity of SidC, which is activated by the lipid molecule, PI(4)P. Furthermore, our results suggest that proper spatial localization of SidC to the cytoplasmic surface of the bacterial phagosome through the binding with PI(4)P is crucial to its function.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
DOI：
10.1073/pnas.1207903109
发表时间：
2012-08-21
影响因子：
11.1
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影响因子：
16
作者：
Kamadurai, Hari B.;Souphron, Judith;Scott, Daniel C.;Duda, David M.;Miller, Darcie J.;Stringer, Daniel;Piper, Robert C.;Schulman, Brenda A.
通讯作者：
Schulman, Brenda A.
影响因子：
4.1
作者：
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DOI：
10.1111/tra.12158
发表时间：
2014-05
期刊：
Traffic (Copenhagen, Denmark)
影响因子：
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作者：
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通讯作者：
Reinisch KM