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- PDB-7dpt: Structural basis for ligand binding modes of CTP synthase -

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Basic information

Entry
Database: PDB / ID: 7dpt
TitleStructural basis for ligand binding modes of CTP synthase
ComponentsCTP synthaseCTP synthetase
KeywordsLIGASE / 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 speciesDrosophila melanogaster (fruit fly)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.48 Å
AuthorsLiu, J.L. / Zhou, X. / Guo, C.J. / Chang, C.C.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: 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.
History
DepositionDec 21, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 15, 2021Provider: repository / Type: Initial release

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Assembly

Deposited unit
A: CTP synthase
B: CTP synthase
C: CTP synthase
D: CTP synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)285,20432
Polymers278,1584
Non-polymers7,04628
Water1,72996
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: homology
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area21950 Å2
ΔGint-178 kcal/mol
Surface area77820 Å2

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Components

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Protein , 1 types, 4 molecules ABCD

#1: Protein
CTP synthase / CTP synthetase / CTP synthetase / UTP--ammonia ligase


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)

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Non-polymers , 6 types, 124 molecules

#2: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


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
#3: Chemical
ChemComp-DON / 6-DIAZENYL-5-OXO-L-NORLEUCINE / (S)-2-AMINO-6-DIAZENYL-5-OXOHEXANOIC ACID


Type: L-peptide linking / Mass: 173.170 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C6H11N3O3 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


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
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-5ZL / [[(2~{R},3~{S},4~{R},5~{R})-3,4-bis(oxidanyl)-5-(2-oxidanyl-4-phosphonooxy-pyrimidin-1-yl)oxolan-2-yl]methoxy-oxidanyl-phosphoryl] phosphono hydrogen phosphate


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 / water / Water


Mass: 18.015 Da / Num. of mol.: 96 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: 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 solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DARK FIELD
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.48 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 107556 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 44.22 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004718320
ELECTRON MICROSCOPYf_angle_d0.668924884
ELECTRON MICROSCOPYf_chiral_restr0.04572780
ELECTRON MICROSCOPYf_plane_restr0.00433160
ELECTRON MICROSCOPYf_dihedral_angle_d17.80652608

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