Crystal structure of Escherichia coli cytidine triphosphate synthetase, a nucleotide-regulated glutamine amidotransferase/ATP-dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets
Crystal structure of Escherichia coli cytidine triphosphate synthetase, a nucleotide-regulated glutamine amidotransferase/ATP-dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets
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DOI：
10.1021/bi0496945
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发表时间：
2004-06-01
期刊：
影响因子：
2.9
通讯作者：
Baldwin, EP
中科院分区：
文献类型：
--
作者：
Endrizzi, JA;Kim, HS;Baldwin, EP
胞苷三磷酸合成酶（Cytidine triphosphate synthetases, ctps）由UTP和谷氨酰胺合成CTP，并通过与四种三磷酸核糖核苷酸的相互作用调节细胞内CTP水平。采用Hg-MAD相法对大肠杆菌CTPS的2.3埃分辨率晶体结构进行了解析。该结构揭示了一个近乎对称的222四聚体，其中每个双功能单体包含一个脱硫生物素合成酶样氨基寡合酶n端结构域和一个1型谷氨酰胺氨基转移酶c端结构域。对于每个酰胺寡聚酶活性位点，必需的ATP和utp结合表面由三个单体组成，这表明活性需要四聚体的形成，并且核苷酸依赖的二聚体-四聚体平衡有助于观察到的正协同性。谷氨酰胺水解酶和酰胺寡聚酶活性位点之间的门控通道跨越25埃，为氨的扩散提供了途径。在与谷氨酰胺水解活性位点相邻的裂缝底部，溶剂可以进入该通道，为外源氨提供了一个入口点。结构相关的GTP酶的鸟嘌呤核苷酸结合位点叠加在这个裂缝上，为GTP的变构调节提供了见解。赋予核苷耐药性和释放CTP抑制的突变映射到靠近utp结合位点的口袋，可以容纳嘧啶环。其位置表明竞争性反馈抑制是通过与底物三磷酸结合位点重叠的不同产物/药物结合位点来影响的。总的来说，大肠杆菌的结构为其他ctps的同源性建模和基于结构的抗ctps疗法设计提供了框架。
Cytidine triphosphate synthetases (CTPSs) produce CTP from UTP and glutamine, and regulate intracellular CTP levels through interactions with the four ribonucleotide triphosphates. We solved the 2.3-Angstrom resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a Type 1 glutamine amidotransferase C-terminal domain. For each amidoligase active site, essential ATP- and UTP-binding surfaces are contributed by three monomers, suggesting that activity requires tetramer formation, and that a nucleotide-dependent dimer-tetramer equilibrium contributes to the observed positive cooperativity. A gated channel that spans 25 Angstrom between the glutamine hydrolysis and amidoligase active sites provides a path for ammonia diffusion. The channel is accessible to solvent at the base of a cleft adjoining the glutamine hydrolysis active site, providing an entry point for exogenous ammonia. Guanine nucleotide binding sites of structurally related GTPases superimpose on this cleft, providing insights into allosteric regulation by GTP. Mutations that confer nucleoside drug resistance and release CTP inhibition map to a pocket that neighbors the UTP-binding site and can accommodate a pyrimidine ring. Its location suggests that competitive feedback inhibition is affected via a distinct product/drug binding site that overlaps the substrate triphosphate binding site. Overall, the E. coli structure provides a framework for homology modeling of other CTPSs and structure-based design of anti-CTPS therapeutics.