Insights into Ubiquitin Product Release in Hydrolysis Catalyzed by the Bacterial Deubiquitinase SdeA.
Insights into Ubiquitin Product Release in Hydrolysis Catalyzed by the Bacterial Deubiquitinase SdeA.
复制标题
对细菌去泛素酶SDEA催化的水解中泛素产物释放的见解。
DOI：
10.1021/acs.biochem.0c00760
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发表时间：
2021-03-02
期刊：
影响因子：
2.9
通讯作者：
Das C
中科院分区：
文献类型：
--
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
我们报道了嗜肺军团菌效应蛋白SdeA的去泛素化酶结构域（SdeADUB）的（催化半胱氨酸）突变为丙氨酸的突变体与其泛素（Ub）产物的共晶体结构。与之前表征的SdeADUB与自杀性抑制剂泛素乙烯基甲酯（Ub - VME）形成的复合物相比，在该产物结合结构中，大多数分子间相互作用得以保留，Ub - VME的结构模拟了酰基酶硫酯中间体。核磁共振滴定研究显示出一种化学位移扰动模式，表明在溶液中同样存在这些相互作用。等温滴定量热法和核磁共振滴定数据表明，野生型SdeADUB对Ub的亲和力明显低于半胱氨酸突变为丙氨酸的突变体。这可能是由于野生型SdeADUB中催化半胱氨酸残基的硫醇阴离子与Ub中C端甘氨酸76残基的羧基之间存在排斥作用。在SdeADUB催化过程中，这种静电排斥在酰基酶中间体中可裂解的异肽键水解以及Ub片段中C端羧基随之形成后产生。我们推测这种静电排斥可能会加速SdeADUB对Ub产物的释放。我们注意到，类似的排斥相互作用也可能发生在其他去泛素化酶以及泛素样蛋白修饰物的水解酶中，并且可能构成该酶家族中一种相当普遍的产物释放机制。对于在酶 - 底物结合过程中形成广泛蛋白质 - 蛋白质相互作用的一类酶而言，这可能是一个潜在的重要特征。
We report the co-crystal structure of the (catalytic Cys)-to-Ala mutant of the deubiquitinase domain of the Legionella pneumophila effector SdeA (SdeADUB) with its ubiquitin (Ub) product. Most of the intermolecular interactions are preserved in this product-bound structure compared to the previously characterized complex of SdeADUB with the suicide inhibitor, ubiquitin vinylmethyl ester (Ub-VME), whose structure models the acyl-enzyme thioester intermediate. NMR titration studies show a chemical shift perturbation pattern that suggests that the same interactions also exist in solution. Isothermal titration calorimetry and NMR titration data reveal that the wild-type SdeADUB has a significantly lower affinity to Ub than the Cys-to-Ala mutant. This is potentially due to repulsive interaction between the thiolate ion of the catalytic Cys residue in WT SdeADUB and the carboxylate group of the C-terminal Gly76 residue in Ub. In the context of SdeADUB catalysis, this electrostatic repulsion arises after the hydrolysis of the scissile isopeptide bond in the acyl-enzyme intermediate and consequent formation of the C-terminal carboxylic group in the Ub fragment. We hypothesize that this electrostatic repulsion may expedite the release of Ub product by SdeADUB. We note that similar repulsive interactions may also occur in other deubiquitinases and hydrolases of ubiquitin-like protein modifiers and may constitute a fairly general mechanism of product release within this family. This is a potentially important feature for a family of enzymes that form extensive protein-protein interactions during enzyme-substrate engagement.
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影响因子：
7.7
作者：
Morrow ME;Morgan MT;Clerici M;Growkova K;Yan M;Komander D;Sixma TK;Simicek M;Wolberger C
通讯作者：
Wolberger C
影响因子：
16.6
作者：
Hermanns T;Pichlo C;Woiwode I;Klopffleisch K;Witting KF;Ovaa H;Baumann U;Hofmann K
通讯作者：
Hofmann K
影响因子：
16
作者：
Kwasna D;Abdul Rehman SA;Natarajan J;Matthews S;Madden R;De Cesare V;Weidlich S;Virdee S;Ahel I;Gibbs-Seymour I;Kulathu Y
通讯作者：
Kulathu Y
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：
10.1107/s0907444909042073
发表时间：
2010-01
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Chen VB;Arendall WB 3rd;Headd JJ;Keedy DA;Immormino RM;Kapral GJ;Murray LW;Richardson JS;Richardson DC
通讯作者：
Richardson DC