School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China.
CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, 200031, China.
Proc Natl Acad Sci U S A. 2021 Jul 27;118(30). doi: 10.1073/pnas.2026621118.
Cytidine triphosphate synthase (CTPS), which comprises an ammonia ligase domain and a glutamine amidotransferase domain, catalyzes the final step of de novo CTP biosynthesis. The activity of CTPS is regulated by the binding of four nucleotides and glutamine. While glutamine serves as an ammonia donor for the ATP-dependent conversion of UTP to CTP, the fourth nucleotide GTP acts as an allosteric activator. Models have been proposed to explain the mechanisms of action at the active site of the ammonia ligase domain and the conformational changes derived by GTP binding. However, actual GTP/ATP/UTP binding modes and relevant conformational changes have not been revealed fully. Here, we report the discovery of binding modes of four nucleotides and a glutamine analog 6-diazo-5-oxo-L-norleucine in CTPS by cryo-electron microscopy with near-atomic resolution. Interactions between GTP and surrounding residues indicate that GTP acts to coordinate reactions at both domains by directly blocking ammonia leakage and stabilizing the ammonia tunnel. Additionally, we observe the ATP-dependent UTP phosphorylation intermediate and determine interacting residues at the ammonia ligase. A noncanonical CTP binding at the ATP binding site suggests another layer of feedback inhibition. Our findings not only delineate the structure of CTPS in the presence of all substrates but also complete our understanding of the underlying mechanisms of the allosteric regulation and CTP synthesis.
三磷酸胞苷合酶（CTPS）由氨连接酶结构域和谷氨酰胺转氨酶结构域组成，催化从头合成 CTP 的最后一步。CTPS 的活性受四个核苷酸和谷氨酰胺的结合调节。虽然谷氨酰胺作为氨供体，参与 UTP 在 ATP 依赖性转化为 CTP 的反应，但第四个核苷酸 GTP 作为别构激活剂。已经提出了模型来解释氨连接酶结构域的活性位点和 GTP 结合引起的构象变化的作用机制。然而，实际的 GTP/ATP/UTP 结合模式和相关的构象变化尚未完全揭示。在这里，我们通过近原子分辨率的冷冻电镜报告了 CTPS 中四个核苷酸和谷氨酰胺类似物 6-重氮-5-氧代-L-正亮氨酸结合模式的发现。GTP 与周围残基的相互作用表明，GTP 通过直接阻断氨泄漏和稳定氨隧道，来协调两个结构域的反应。此外，我们观察到氨连接酶上的 ATP 依赖性 UTP 磷酸化中间产物和相互作用的残基。在 ATP 结合位点的非典型 CTP 结合表明了另一个反馈抑制的层次。我们的发现不仅描绘了所有底物存在下 CTPS 的结构，而且还完善了我们对别构调节和 CTP 合成的潜在机制的理解。
