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- PDB-7mh1: Human CTPS2 bound to CTP -

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Basic information

Entry
Database: PDB / ID: 7mh1
TitleHuman CTPS2 bound to CTP
ComponentsCTP synthase 2
KeywordsLIGASE / glutaminase / amidoligase / nucleotide metabolism
Function / homology
Function and homology information


cytoophidium / CTP synthase (glutamine hydrolysing) / CTP synthase activity / pyrimidine nucleotide metabolic process / 'de novo' CTP biosynthetic process / pyrimidine nucleobase biosynthetic process / Interconversion of nucleotide di- and triphosphates / CTP biosynthetic process / mitochondrion / ATP binding ...cytoophidium / CTP synthase (glutamine hydrolysing) / CTP synthase activity / pyrimidine nucleotide metabolic process / 'de novo' CTP biosynthetic process / pyrimidine nucleobase biosynthetic process / Interconversion of nucleotide di- and triphosphates / CTP biosynthetic process / mitochondrion / ATP binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
CTP synthase / CTP synthase, N-terminal / CTP synthase GATase domain / CTP synthase N-terminus / Glutamine amidotransferase / Glutamine amidotransferase class-I / Glutamine amidotransferase type 1 domain profile. / Class I glutamine amidotransferase (GATase) domain / Class I glutamine amidotransferase-like / P-loop containing nucleotide triphosphate hydrolases ...CTP synthase / CTP synthase, N-terminal / CTP synthase GATase domain / CTP synthase N-terminus / Glutamine amidotransferase / Glutamine amidotransferase class-I / Glutamine amidotransferase type 1 domain profile. / Class I glutamine amidotransferase (GATase) domain / Class I glutamine amidotransferase-like / P-loop containing nucleotide triphosphate hydrolases / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
CYTIDINE-5'-TRIPHOSPHATE / CTP synthase 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsLynch, E.M. / Kollman, J.M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM118396 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Structural basis for isoform-specific inhibition of human CTPS1.
Authors: Eric M Lynch / Michael A DiMattia / Steven Albanese / Gydo C P van Zundert / Jesse M Hansen / Joel D Quispe / Madison A Kennedy / Andreas Verras / Kenneth Borrelli / Angela V Toms / Neelu ...Authors: Eric M Lynch / Michael A DiMattia / Steven Albanese / Gydo C P van Zundert / Jesse M Hansen / Joel D Quispe / Madison A Kennedy / Andreas Verras / Kenneth Borrelli / Angela V Toms / Neelu Kaila / Kevin D Kreutter / Joshua J McElwee / Justin M Kollman /
Abstract: Cytidine triphosphate synthase 1 (CTPS1) is necessary for an effective immune response, as revealed by severe immunodeficiency in CTPS1-deficient individuals [E. Martin ], [] [510], [288-292] ([2014]) ...Cytidine triphosphate synthase 1 (CTPS1) is necessary for an effective immune response, as revealed by severe immunodeficiency in CTPS1-deficient individuals [E. Martin ], [] [510], [288-292] ([2014]). CTPS1 expression is up-regulated in activated lymphocytes to expand CTP pools [E. Martin ], [] [510], [288-292] ([2014]), satisfying increased demand for nucleic acid and lipid synthesis [L. D. Fairbanks, M. Bofill, K. Ruckemann, H. A. Simmonds], [ ] [270], [29682-29689] ([1995]). Demand for CTP in other tissues is met by the CTPS2 isoform and nucleoside salvage pathways [E. Martin ], [] [510], [288-292] ([2014]). Selective inhibition of the proliferative CTPS1 isoform is therefore desirable in the treatment of immune disorders and lymphocyte cancers, but little is known about differences in regulation of the isoforms or mechanisms of known inhibitors. We show that CTP regulates both isoforms by binding in two sites that clash with substrates. CTPS1 is less sensitive to CTP feedback inhibition, consistent with its role in increasing CTP levels in proliferation. We also characterize recently reported small-molecule inhibitors, both CTPS1 selective and nonselective. Cryo-electron microscopy (cryo-EM) structures reveal these inhibitors mimic CTP binding in one inhibitory site, where a single amino acid substitution explains selectivity for CTPS1. The inhibitors bind to CTPS assembled into large-scale filaments, which for CTPS1 normally represents a hyperactive form of the enzyme [E. M. Lynch ], [] [24], [507-514] ([2017]). This highlights the utility of cryo-EM in drug discovery, particularly for cases in which targets form large multimeric assemblies not amenable to structure determination by other techniques. Both inhibitors also inhibit the proliferation of human primary T cells. The mechanisms of selective inhibition of CTPS1 lay the foundation for the design of immunosuppressive therapies.
History
DepositionApr 14, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 13, 2021Provider: repository / Type: Initial release
Revision 1.1May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Assembly

Deposited unit
H: CTP synthase 2
J: CTP synthase 2
K: CTP synthase 2
L: CTP synthase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)267,09720
Polymers263,0384
Non-polymers4,06016
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
CTP synthase 2 / CTP synthetase 2 / UTP--ammonia ligase 2


Mass: 65759.430 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CTPS2 / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: Q9NRF8, CTP synthase (glutamine hydrolysing)
#2: Chemical
ChemComp-CTP / CYTIDINE-5'-TRIPHOSPHATE


Mass: 483.156 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C9H16N3O14P3
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN

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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: CTPS2 tetramer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.9
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: BRIGHT FIELD
Image recordingElectron dose: 90 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2Leginonimage acquisition
4CTFFIND4CTF correction
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 46236 / Symmetry type: POINT

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