|Entry||Database: PDB / ID: 7dpt|
|Title||Structural basis for ligand binding modes of CTP synthase|
|Components||CTP synthaseCTP synthetase|
|Keywords||LIGASE / substrate-bound / filament / DON|
|Function / homology|
Function and homology information
Interconversion of nucleotide di- and triphosphates / larval lymph gland hemopoiesis / CTP synthase activity / cytoophidium / CTP synthase (glutamine hydrolysing) / 'de novo' CTP biosynthetic process / pyrimidine nucleobase biosynthetic process / CTP biosynthetic process / glutamine metabolic process / ATP binding ...Interconversion of nucleotide di- and triphosphates / larval lymph gland hemopoiesis / CTP synthase activity / cytoophidium / CTP synthase (glutamine hydrolysing) / 'de novo' CTP biosynthetic process / pyrimidine nucleobase biosynthetic process / CTP biosynthetic process / glutamine metabolic process / ATP binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
CTP synthase GATase domain / CTP synthase, N-terminal / CTP synthase / CTP synthase N-terminus / Glutamine amidotransferase type 1 domain profile. / Glutamine amidotransferase class-I / Glutamine amidotransferase / Class I glutamine amidotransferase-like / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Chem-5ZL / ADENOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / 6-DIAZENYL-5-OXO-L-NORLEUCINE / CTP synthase
Similarity search - Component
|Biological species||Drosophila melanogaster (fruit fly)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.48 Å|
|Authors||Liu, J.L. / Zhou, X. / Guo, C.J. / Chang, C.C.|
|Funding support|| China, 1items |
|Citation||Journal: Proc Natl Acad Sci U S A / Year: 2021|
Title: Structural basis for ligand binding modes of CTP synthase.
Authors: Xian Zhou / Chen-Jun Guo / Chia-Chun Chang / Jiale Zhong / Huan-Huan Hu / Guang-Ming Lu / Ji-Long Liu /
Abstract: 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 ...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.
|Structure viewer||Molecule: |
Downloads & links
A: CTP synthase
B: CTP synthase
C: CTP synthase
D: CTP synthase
-Protein , 1 types, 4 molecules A
B C D
Mass: 69539.453 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: CTPsyn, CG45070
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q9VUL1, CTP synthase (glutamine hydrolysing)
-Non-polymers , 6 types, 124 molecules
Mass: 523.180 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
Type: L-peptide linking / Mass: 173.170 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C6H11N3O3 / Feature type: SUBJECT OF INVESTIGATION
Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Mass: 24.305 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
ChemComp-5ZL / [[(
Mass: 565.129 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: C9H17N2O18P4 / Feature type: SUBJECT OF INVESTIGATION
|#7: Water|| ChemComp-HOH / |
|Has ligand of interest||Y|
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction|
|Component||Name: Drosophila substrate-bound CTP synthase / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT|
|Source (natural)||Organism: Drosophila melanogaster (fruit fly)|
|Source (recombinant)||Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)|
|Buffer solution||pH: 7.5|
|Specimen||Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: DARK FIELD|
|Image recording||Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k)|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Symmetry||Point symmetry: D2 (2x2 fold dihedral)|
|3D reconstruction||Resolution: 2.48 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 107556 / Symmetry type: POINT|
|Refinement||Cross valid method: NONE|
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
|Displacement parameters||Biso mean: 44.22 Å2|
|Refine LS restraints|
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