Cocrystallization of ubiquitin-deubiquitinase complexes through disulfide linkage.
Cocrystallization of ubiquitin-deubiquitinase complexes through disulfide linkage.
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DOI：
10.1107/s2059798323008501
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发表时间：
2023-11-01
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影响因子：
--
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中科院分区：
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两种通过二硫键连接的与泛素结合的细菌去泛素化酶的晶体结构揭示了泛素的结合模式。这些结构表明，二硫键连接是捕获共价泛素 - 去泛素化酶复合物结构的一种有效策略。
去泛素化酶（DUBs）对泛素（Ub）识别的结构表征在很大程度上依赖于DUB通过其催化半胱氨酸与泛素C端亲电体的共价络合。泛素亲电体可通过内含肽化学结合化学合成获得。在此，研究人员探讨是否可以使用一种将泛素中最后一个甘氨酸替换为半胱氨酸的泛素半胱氨酸突变体，通过更简单的二硫键化学来获得DUB - Ub共价复合物。该泛素半胱氨酸突变体在一系列真核和原核DUBs中形成二硫键的情况差异很大，有些未检测到反应，而另一些则能大量生成二硫键复合物。利用这种方法，两种通过二硫键连接的与泛素结合的复合物得以结晶，一种涉及嗜肺军团菌效应蛋白SdeA DUB，另一种涉及东方体效应蛋白OtDUB。此前，这两种DUBs分别通过C端亲电体策略和非共价络合以与泛素结合的形式结晶。正如预期的那样，二硫键连接的SdeA DUB - Ub复合物结晶，而在OtDUB复合物中，与泛素突变体形成二硫键的半胱氨酸并非催化半胱氨酸。与SdeA DUB催化半胱氨酸形成二硫键时，携带活性位点半胱氨酸的螺旋会发生局部扭曲，而OtDUB则利用一个表面暴露的半胱氨酸与泛素突变体发生反应。
The crystal structures of two disulfide-linked ubiquitin-bound bacterial deubiquitinases reveal the mode of ubiquitin binding. These structures show that disulfide linking is an effective strategy for capturing covalent ubiquitin–deubiquitinase complex structures. Structural characterization of the recognition of ubiquitin (Ub) by deubiquitinases (DUBs) has largely relied on covalent complexation of the DUB through its catalytic cysteine with a Ub C-terminal electrophile. The Ub electrophiles are accessed through intein chemistry in conjunction with chemical synthesis. Here, it was asked whether DUB–Ub covalent complexes could instead be accessed by simpler disulfide chemistry using a Ub cysteine mutant in which the last glycine has been replaced with a cysteine. The Ub cysteine mutant displayed a wide variability in disulfide formation across a panel of eukaryotic and prokaryotic DUBs, with some showing no detectable reaction while others robustly produced a disulfide complex. Using this approach, two disulfide-linked ubiquitin-bound complexes were crystallized, one involving the Legionella pneumophila effector SdeA DUB and the other involving the Orientia effector OtDUB. These DUBs had previously been crystallized in Ub-bound forms using the C-terminal electrophile strategy and noncovalent complexation, respectively. While the disulfide-linked SdeA DUB–Ub complex crystallized as expected, in the OtDUB complex the disulfide bond to the Ub mutant involved a cysteine that differed from the catalytic cysteine. Disulfide formation with the SdeA DUB catalytic cysteine was accompanied by local distortion of the helix carrying the active-site cysteine, whereas OtDUB reacted with the Ub mutant using a surface-exposed cysteine.
对细菌去泛素酶SDEA催化的水解中泛素产物释放的见解。
影响因子：
2.9
作者：
Sheedlo MJ;Kenny S;Podkorytov IS;Brown K;Ma J;Iyer S;Hewitt CS;Arbough T;Mikhailovskii O;Flaherty DP;Wilson MA;Skrynnikov NR;Das C
通讯作者：
Das C