Structure and Functional Characterization of Human Aspartate Transcarbamoylase, the Target of the Anti-tumoral Drug PALA
Structure and Functional Characterization of Human Aspartate Transcarbamoylase, the Target of the Anti-tumoral Drug PALA
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DOI：
10.1016/j.str.2016.05.001
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发表时间：
2016-07-06
期刊：
影响因子：
5.7
通讯作者：
Ramon-Maiques, Santiago
中科院分区：
文献类型：
--
作者：
Ruiz-Ramos, Alba;Velazquez-Campoy, Adrian;Ramon-Maiques, Santiago
CAD是一种启动和控制动物体内嘧啶从头合成的多酶蛋白，通过其天冬氨酸转甲氨基酰化酶（ATCase）结构域结合成1.5 MDa的颗粒。尽管原核生物的ATCase结构众多，但我们缺乏CAD的ATCase域的结构信息。在这里，我们报告了人类ATCase的结构和功能特征，证实了与细菌同源物的总体相似性。出乎意料的是，人类ATCase表现出协同效应，降低了抗肿瘤药物PALA的亲和力。结合结构、诱变和生化分析，我们确定了导致亚基之间协同作用的构象变化的必要调控和传递的关键因素。其中一种基因R2024的突变最近被发现导致了第一个非致命性的CAD缺陷。我们在人类ATCase中复制了这种突变，并测量了其影响，证明这种精氨酸是调节配体低亲和力和高亲和力状态平衡的分子开关的一部分。
CAD, the multienzymatic protein that initiates and controls de novo synthesis of pyrimidines in animals, associates through its aspartate transcarbamoylase (ATCase) domain into particles of 1.5 MDa. Despite numerous structures of prokaryotic ATCases, we lack structural information on the ATCase domain of CAD. Here, we report the structure and functional characterization of human ATCase, confirming the overall similarity with bacterial homologs. Unexpectedly, human ATCase exhibits cooperativity effects that reduce the affinity for the anti-tumoral drug PALA. Combining structural, mutagenic, and biochemical analysis, we identified key elements for the necessary regulation and transmission of conformational changes leading to cooperativity between subunits. Mutation of one of these elements, R2024, was recently found to cause the first non-lethal CAD deficit. We reproduced this mutation in human ATCase and measured its effect, demonstrating that this arginine is part of a molecular switch that regulates the equilibrium between low- and high-affinity states for the ligands.