Role of Conserved Histidine and Serine in the HCXXXXXRS Motif of Human Dual-Specificity Phosphatase 5.
Role of Conserved Histidine and Serine in the HCXXXXXRS Motif of Human Dual-Specificity Phosphatase 5.
复制标题
人双特异性磷酸酶 5 的 HCXXXXXRS 基序中保守的组氨酸和丝氨酸的作用。
DOI：
10.1021/acs.jcim.8b00919
复制
发表时间：
2019
影响因子：
5.6
通讯作者：
Talipov,MaratR
中科院分区：
文献类型：
--
作者：
Gupta,Ankan;Brahmbhatt,Jaladhi;Syrlybaeva,Raulia;Bodnar,Catherine;Bodnar,Natalia;Bongard,Robert;Pokkuluri,PhaniRaj;Sem,DanielS;Ramchandran,Ramani;Rathore,Rajendra;Talipov,MaratR
背景丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）信号通路在维持机体生理平衡和正常组织发育中具有重要的功能。该途径受到严格调控，其部分由双特异性磷酸酶（DUSPs）介导，所述双特异性磷酸酶使ERK蛋白家族的丝氨酸、苏氨酸和酪氨酸残基去磷酸化。DUSP 5具有很高的临床意义，因为我们在血管异常患者中发现了这种蛋白质的突变。与其他DUSPs不同，DUSP 5对底物pERK 1/2具有独特的特异性。使用分子对接和模拟策略，我们先前表明DUSP 5有两个口袋，它们以特定的方式用于促进其底物pERK 1/2催化的特异性。值得注意的是，大多数DUSPs在其催化位点上具有高度相似性。研究DUSP 5的催化结构域，并确定对pERK 1/2去磷酸化起重要作用的氨基酸残基，对于开发DUSP 5靶向治疗的小分子药物具有重要意义。结果本研究利用计算机模拟技术确定并预测了DUSP 5催化结构域中两个保守氨基酸残基H262和S270的重要性。模型研究预测，如果这些关键的保守残基发生突变，DUSP 5的催化活性将发生改变。我们接下来产生了独立的谷胱甘肽-S-转移酶（GST）标记的全长DUSP 5突变体蛋白，其在磷酸酶结构域中携带特异性突变H262 F和S270 A。对这些纯化的蛋白质进行生化分析，与我们的计算预测一致，当与野生型GST-1相比时，我们观察到具有合成小分子底物（pNPP）和生理相关底物（pERK）的两种突变体的酶活性动力学曲线改变。DUSP 5蛋白。结论我们的分子模拟和生物化学研究表明，磷酸酶的酶活性可以通过突变来操纵，催化位点（磷酸酶结构域）中的特定保守氨基酸残基。该策略可以促进将用作DUSP 5活性的激动剂/拮抗剂的小分子的产生。
BackgroundThe mitogen-activated protein kinase (MAPK) pathway is functionally generic and critical in maintaining physiological homeostasis and normal tissue development. This pathway is under tight regulation, which is in part mediated by dual-specific phosphatases (DUSPs), which dephosphorylate serine, threonine, and tyrosine residues of the ERK family of proteins. DUSP5 is of high clinical interest because of mutations we identified in this protein in patients with vascular anomalies. Unlike other DUSPs, DUSP5 has unique specificity toward substrate pERK1/2. Using molecular docking and simulation strategies, we previously showed that DUSP5 has two pockets, which are utilized in a specific fashion to facilitate specificity toward catalysis of its substrate pERK1/2. Remarkably, most DUSPs share high similarity in their catalytic sites. Studying the catalytic domain of DUSP5 and identifying amino acid residues that are important for dephosphorylating pERK1/2 could be critical in developing small molecules for therapies targeting DUSP5.ResultsIn this study, we utilized computational modeling to identify and predict the importance of two conserved amino acid residues, H262 and S270, in the DUSP5 catalytic site. Modeling studies predicted that catalytic activity of DUSP5 would be altered if these critical conserved residues were mutated. We next generated independent Glutathione-S-Transferase (GST)-tagged full-length DUSP5 mutant proteins carrying specific mutations H262F and S270A in the phosphatase domain. Biochemical analysis was performed on these purified proteins, and consistent with our computational prediction, we observed altered enzyme activity kinetic profiles for both mutants with a synthetic small molecule substrate (pNPP) and the physiological relevant substrate (pERK) when compared to wild type GST-DUSP5 protein.ConclusionOur molecular modeling and biochemical studies combined demonstrate that enzymatic activity of phosphatases can be manipulated by mutating specific conserved amino acid residues in the catalytic site (phosphatase domain). This strategy could facilitate generation of small molecules that will serve as agonists/antagonists of DUSP5 activity.