Convergent Evolution in the Assembly of Polyubiquitin Degradation Signals by the Shigella flexneri IpaH9.8 Ligase
Convergent Evolution in the Assembly of Polyubiquitin Degradation Signals by the Shigella flexneri IpaH9.8 Ligase
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DOI：
10.1074/jbc.m114.609164
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发表时间：
2014-12-05
影响因子：
4.8
通讯作者：
Haas, Arthur L.
中科院分区：
文献类型：
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作者：
Edwards, Daniel J.;Streich, Frederick C., Jr.;Haas, Arthur L.
背景：IpaH细菌泛素连接酶与真核生物连接酶没有同源性，其作用机制尚不清楚。结果：IpaH9.8是一个协同的变构二聚体，每个亚基上有两个Ubc 5与泛素结合位点。结论：IpaH和真核细胞HECT连接酶之间的动力学相似性表明收敛催化循环进化。重要性：这些是IpaH酶催化机制的第一个机械细节。人类病原体福氏志贺菌通过III型分泌系统部署一组效应蛋白来破坏宿主功能和防御。IpaH家族的10个这样的效应物模拟泛素连接酶，但没有序列或结构同源性，其真核对应物。使用I-125-多聚泛素链形成的速率作为功能读数，IpaH 9.8在纳摩尔范围内（[S]（1/2）= 140 +/- 32 nm; n = 1.8 +/- 0.1）和微摩尔浓度下的协同底物抑制（[S]（1/2）= 740 +/- 240 nm; n = 1.7 +/- 0.2），需要有序结合到每个亚基的两个功能不同的位点。电子等排底物类似物Ubc 5 BC 85 S-泛素氧酯作为野生型Ubc 5 B的竞争性抑制剂，接近I-125-泛素硫酯（Ki = 117 +/- 29 nm），而Ubc 5 BC 85 A产物类似物显示非竞争性抑制（Ki = 2.2 +/- 0.5 nm），与双位点模型一致。相关IpaH 3晶体结构的重新评估（PDB条目3CVR）确定了与观察到的协同性一致的对称二聚体。预测的IpaH9.8二聚体界面的遗传破坏降低了溶液的分子量，并显著消除了多聚泛素链形成的k（cat），但没有消除[S]（1/2）。其他研究表明，协同性需要N-末端富含亮氨酸的重复靶向结构域，并通过Phe转导（395）。此外，这些机械特征在远亲SspH 2肠道沙门氏菌连接酶中是保守的。IpaH 9.8和最近修订的E6 AP/UBE 3A机制之间的动力学相似性（Ronchi，V. P.，克莱因，J.M.，和哈斯，A. L.（2013）E6 AP/UBE 3A泛素连接酶具有两个与泛素结合位点近似的E2。J.Biol.Chem.288，10349-10360）提出了原核和真核连接酶的催化机制的趋同进化。
Background: IpaH bacterial ubiquitin ligases show no homology with eukaryotic ligases, and their mechanism is speculative. Results: IpaH9.8 functions as a cooperative allosteric dimer with two Ubc5 approximate to ubiquitin binding sites per subunit. Conclusion: Kinetic parallels between IpaH and eukaryotic HECT ligases suggest convergent catalytic cycle evolution. Significance: These are the first mechanistic details of the IpaH enzyme catalytic mechanism.The human pathogen Shigella flexneri subverts host function and defenses by deploying a cohort of effector proteins via a type III secretion system. The IpaH family of 10 such effectors mimics ubiquitin ligases but bears no sequence or structural homology to their eukaryotic counterpoints. Using rates of I-125-polyubiquitin chain formation as a functional read out, IpaH9.8 displays V-type positive cooperativity with respect to varying concentrations of its Ubc5B approximate to I-125-ubiquitin thioester co-substrate in the nanomolar range ([S](1/2) = 140 +/- 32 nm; n = 1.8 +/- 0.1) and cooperative substrate inhibition at micromolar concentrations ([S](1/2) = 740 +/- 240 nm; n = 1.7 +/- 0.2), requiring ordered binding to two functionally distinct sites per subunit. The isosteric substrate analog Ubc5BC85S-ubiquitin oxyester acts as a competitive inhibitor of wild-type Ubc5B approximate to I-125-ubiquitin thioester (K-i = 117 +/- 29 nm), whereas a Ubc5BC85A product analog shows noncompetitive inhibition (K-i = 2.2 +/- 0.5 m), consistent with the two-site model. Re-evaluation of a related IpaH3 crystal structure (PDB entry 3CVR) identifies a symmetric dimer consistent with the observed cooperativity. Genetic disruption of the predicted IpaH9.8 dimer interface reduces the solution molecular weight and significantly ablates the k(cat) but not [S](1/2) for polyubiquitin chain formation. Other studies demonstrate that cooperativity requires the N-terminal leucine-rich repeat-targeting domain and is transduced through Phe(395). Additionally, these mechanistic features are conserved in a distantly related SspH2 Salmonella enterica ligase. Kinetic parallels between IpaH9.8 and the recently revised mechanism for E6AP/UBE3A (Ronchi, V. P., Klein, J. M., and Haas, A. L. (2013) E6AP/UBE3A ubiquitin ligase harbors two E2 approximate to ubiquitin binding sites. J. Biol. Chem. 288, 10349-10360) suggest convergent evolution of the catalytic mechanisms for prokaryotic and eukaryotic ligases.