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The Importance of Charge in Perturbing the Aromatic Glue Stabilizing the Protein–Protein Interface of Homodimeric tRNA-Guanine Transglycosylase
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2020-11-09 , DOI: 10.1021/acschembio.0c00700 Andreas Nguyen 1 , Dzung Nguyen 1 , Tran Xuan Phong Nguyen 1 , Maurice Sebastiani 1 , Stefanie Dörr 1 , Oscar Hernandez-Alba 2 , François Debaene 2 , Sarah Cianférani 2 , Andreas Heine 1 , Gerhard Klebe 1 , Klaus Reuter 1
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2020-11-09 , DOI: 10.1021/acschembio.0c00700 Andreas Nguyen 1 , Dzung Nguyen 1 , Tran Xuan Phong Nguyen 1 , Maurice Sebastiani 1 , Stefanie Dörr 1 , Oscar Hernandez-Alba 2 , François Debaene 2 , Sarah Cianférani 2 , Andreas Heine 1 , Gerhard Klebe 1 , Klaus Reuter 1
Affiliation
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Bacterial tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the modified tRNA nucleoside queuosine present in the anticodon wobble position of tRNAs specific for aspartate, asparagine, histidine, and tyrosine. Inactivation of the tgt gene leads to decreased pathogenicity of Shigella bacteria. Therefore, Tgt constitutes a putative target for Shigellosis drug therapy. Since it is only active as homodimer, interference with dimer-interface formation may, in addition to active-site inhibition, provide further means to disable this protein. A cluster of four aromatic residues seems important to stabilize the homodimer. We mutated residues of this aromatic cluster and analyzed each mutated variant with respect to the dimer and thermal stability or enzyme activity by applying native mass spectrometry, a thermal shift assay, enzyme kinetics, and X-ray crystallography. Our structural studies indicate a strong influence of pH on the homodimer stability. Apparently, protonation of a histidine within the aromatic cluster supports the collapse of an essential structural motif within the dimer interface at slightly acidic pH.
中文翻译：
电荷在稳定稳定二聚体tRNA-鸟嘌呤转糖基化酶的蛋白质-蛋白质界面的芳香胶中的重要性。
细菌tRNA-鸟嘌呤转糖基化酶（Tgt）参与修饰的tRNA核苷queuosine的生物合成，该修饰的tRNA在对天门冬氨酸，天冬酰胺，组氨酸和酪氨酸特异的tRNA的反密码子摆动位置中存在。tgt基因的失活导致志贺氏菌的致病性降低菌。因此，Tgt构成了志贺氏菌药物治疗的假定靶标。由于它仅作为同型二聚体具有活性，因此除抑制活性位点外，对二聚体界面形成的干扰还可以提供进一步的手段来使该蛋白质失活。四个芳族残基簇似乎对稳定同型二聚体很重要。我们突变了该芳香族簇的残基，并通过应用天然质谱，热位移分析，酶动力学和X射线晶体学分析了每个突变体的二聚体和热稳定性或酶活性。我们的结构研究表明，pH值对同型二聚体的稳定性有很大影响。显然，芳香族簇中组氨酸的质子化支持在弱酸性pH下二聚体界面内基本结构基序的崩溃。
更新日期：2020-11-21
中文翻译：
电荷在稳定稳定二聚体tRNA-鸟嘌呤转糖基化酶的蛋白质-蛋白质界面的芳香胶中的重要性。
细菌tRNA-鸟嘌呤转糖基化酶（Tgt）参与修饰的tRNA核苷queuosine的生物合成，该修饰的tRNA在对天门冬氨酸，天冬酰胺，组氨酸和酪氨酸特异的tRNA的反密码子摆动位置中存在。tgt基因的失活导致志贺氏菌的致病性降低菌。因此，Tgt构成了志贺氏菌药物治疗的假定靶标。由于它仅作为同型二聚体具有活性，因此除抑制活性位点外，对二聚体界面形成的干扰还可以提供进一步的手段来使该蛋白质失活。四个芳族残基簇似乎对稳定同型二聚体很重要。我们突变了该芳香族簇的残基，并通过应用天然质谱，热位移分析，酶动力学和X射线晶体学分析了每个突变体的二聚体和热稳定性或酶活性。我们的结构研究表明，pH值对同型二聚体的稳定性有很大影响。显然，芳香族簇中组氨酸的质子化支持在弱酸性pH下二聚体界面内基本结构基序的崩溃。
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