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首页> 外文期刊>Biochemistry >Contribution of Active Site Residues to Substrate Hydrolysis by USP2: Insights into Catalysis by Ubiquitin Specific Proteases
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Contribution of Active Site Residues to Substrate Hydrolysis by USP2: Insights into Catalysis by Ubiquitin Specific Proteases

机译：活性位点残基对USP2水解底物的贡献：泛素特异性蛋白酶催化的见解

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The ubiquitin-specific protease (USP) structural class represents thenlargest and most diverse family of deubiquitinating enzymes (DUBs). Many USPsnassume important biological roles and emerge as potential targets for therapeuticnintervention. A clear understanding of USP catalytic mechanism requires a func-ntional evaluation of the proposed key active site residues. Crystallographic data ofnubiquitin aldehyde adducts of USP catalytic cores provided structural details on thencatalytic triad residues, namely the conserved Cys and His, and a variable putativenthird residue, and inferred indirect structural roles for two other conserved residuesn(Asn and Asp), in stabilizing via a bridging water molecule the oxyanion of thentetrahedral intermediate (TI).We have expressed the catalytic domain of USP2 andnprobed by site-directed mutagenesis the role of these active site residues in thenhydrolysis of peptide and isopeptide substrates, including a synthetic K48-linkedndiubiquitin substrate for which a label-free, mass spectrometry based assay has beenndeveloped to monitor cleavage. Hydrolysis of ubiquitin-AMC, a model substrate, was not affected by the mutations. Molecularndynamics simulations of USP2, free and complexed with the TI of a bona fide isopeptide substrate, were carried out.We found thatnAsn271 is structurally poised to directly stabilize the oxyanion developed in the acylation step, while being structurally supported bynthe adjacent absolutely conserved Asp575. Mutagenesis data functionally confirmed this structural role independent of the naturen(isopeptide vs peptide) of the bond being cleaved. We also found that Asn574, structurally located as the third member of thencatalytic triad, does not fulfill this role functionally. A dual supporting role is inferred fromdouble-pointmutation and structural datanfor the absolutely conserved residue Asp575, in oxyanion hole formation, and in maintaining the correct alignment and protonationnof His557 for catalytic competency.
机译：泛素特异性蛋白酶（USP）的结构类别代表了最大，最多样化的去泛素化酶（DUBs）家族。许多USPsnas承担着重要的生物学作用，并成为治疗干预的潜在靶标。对USP催化机理的清楚理解需要对提议的关键活性位点残基进行功能评估。 USP催化核的泛素醛加合物的晶体学数据提供了有关催化三联体残基（即保守的Cys和His和可变的推定的第三个残基）的结构细节，并推断了另外两个保守残基n（Asn和Asp）的间接结构作用，通过我们已经表达了USP2的催化结构域，并通过定点诱变探讨了这些活性位点残基在随后水解肽和异肽底物（包括合成的K48连接的二泛素底物）中的作用。已经开发了无标记的基于质谱的测定法来监测裂解。突变不会影响泛素-AMC（一种模型底物）的水解。进行了USP2的分子动力学模拟，该USP2游离并与真正的异肽底物的TI络合。我们发现，nAsn271在结构上可以直接稳定酰化步骤中产生的氧阴离子，同时在结构上受到相邻的绝对保守的Asp575的支持。诱变数据在功能上证实了这种结构性作用，而与所裂解键的性质（异肽对肽）无关。我们还发现，Asn574在结构上是当时催化三联体的第三个成员，并未在功能上实现该角色。从双点突变和绝对保守的残基Asp575的结构数据推断出双重支持作用，包括形成氧阴离子孔，以及保持His557的正确排列和质子化以促进催化能力。

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