Structural basis for ligand binding modes of CTP synthase
| Autor: | Guang-Ming Lu, Jiale Zhong, Xian Zhou, Ji-Long Liu, Huan-Huan Hu, Chen-Jun Guo, Chia-Chun Chang |
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| Rok vydání: | 2021 |
| Předmět: |
GTP'
Protein Conformation Glutamine Allosteric regulation Uridine Triphosphate Ligands Biochemistry Catalysis 03 medical and health sciences 0302 clinical medicine Adenosine Triphosphate Ammonia Animals heterocyclic compounds CTP binding Carbon-Nitrogen Ligases Binding site 030304 developmental biology Glutamine amidotransferase CTP synthase chemistry.chemical_classification 0303 health sciences DNA ligase Multidisciplinary Binding Sites Hydrolysis cytoophidium Cryoelectron Microscopy Biological Sciences allosteric regulation Kinetics Drosophila melanogaster chemistry Biophysics Cytoophidium cryo–electron microscopy 030217 neurology & neurosurgery UTP binding |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America |
| ISSN: | 1091-6490 |
| Popis: | Significance In the current study, we successfully push the resolution to near-atomic levels (2.48 Å) to analyze the polymer structure of Drosophila melanogaster cytidine triphosphate synthase (dmCTPS) with all its substrates (6-diazo-5-oxo-L-norleucine being used to represent glutamine). We have precisely located all ligands in a CTPS structure with a solid electron density map. With this model, we present a structural conformation of the GTP binding site and demonstrate its roles in mediating glutamine binding, NH3 transport, and stabilizing the ammonia tunnel. Additionally, the intermediate in the ATP-dependent phosphorylation reaction is observed allowing us to identify the residues participating in catalysis. 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 Drosophila 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. |
| Databáze: | OpenAIRE |
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