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- EMDB-23833: Human CTPS2 bound to CTP -

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Basic information

Entry
Database: EMDB / ID: EMD-23833
TitleHuman CTPS2 bound to CTP
Map dataHuman CTPS2 bound to CTP
Sample
  • Complex: CTPS2 tetramer
    • Protein or peptide: CTP synthase 2
  • Ligand: CYTIDINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
Keywordsglutaminase / amidoligase / nucleotide metabolism / LIGASE
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 / glutamine metabolic process / 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 / glutamine metabolic process / 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-like / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsLynch EM / Kollman JM
Funding support United States, 1 items
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, 2021-
Header (metadata) releaseOct 13, 2021-
Map releaseOct 13, 2021-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.78
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.78
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7mh1
  • Surface level: 0.78
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23833.png

Downloads & links

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Map

FileDownload / File: emd_23833.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman CTPS2 bound to CTP
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.05 Å/pix.
x 300 pix.
= 315. Å		1.05 Å/pix.
x 300 pix.
= 315. Å		1.05 Å/pix.
x 300 pix.
= 315. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.78 / Movie #1: 0.78
Minimum - Maximum-7.312016 - 13.354913
Average (Standard dev.)0.000000000000356 (±0.2120993)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 315.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z315.000315.000315.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-7.31213.3550.000

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Supplemental data

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Sample components

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Entire : CTPS2 tetramer

EntireName: CTPS2 tetramer
Components
  • Complex: CTPS2 tetramer
    • Protein or peptide: CTP synthase 2
  • Ligand: CYTIDINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: CTPS2 tetramer

SupramoleculeName: CTPS2 tetramer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: CTP synthase 2

MacromoleculeName: CTP synthase 2 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: CTP synthase (glutamine hydrolysing)
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 65.75943 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MKYILVTGGV ISGIGKGIIA SSIGTILKSC GLRVTAIKID PYINIDAGTF SPYEHGEVFV LNDGGEVDLD LGNYERFLDI NLYKDNNIT TGKIYQHVIN KERRGDYLGK TVQVVPHITD AVQEWVMNQA KVPVDGNKEE PQICVIELGG TIGDIEGMPF V EAFRQFQF ...String:
MKYILVTGGV ISGIGKGIIA SSIGTILKSC GLRVTAIKID PYINIDAGTF SPYEHGEVFV LNDGGEVDLD LGNYERFLDI NLYKDNNIT TGKIYQHVIN KERRGDYLGK TVQVVPHITD AVQEWVMNQA KVPVDGNKEE PQICVIELGG TIGDIEGMPF V EAFRQFQF KAKRENFCNI HVSLVPQLSA TGEQKTKPTQ NSVRALRGLG LSPDLIVCRS STPIEMAVKE KISMFCHVNP EQ VICIHDV SSTYRVPVLL EEQSIVKYFK ERLHLPIGDS ASNLLFKWRN MADRYERLQK ICSIALVGKY TKLRDCYASV FKA LEHSAL AINHKLNLMY IDSIDLEKIT ETEDPVKFHE AWQKLCKADG ILVPGGFGIR GTLGKLQAIS WARTKKIPFL GVCL GMQLA VIEFARNCLN LKDADSTEFR PNAPVPLVID MPEHNPGNLG GTMRLGIRRT VFKTENSILR KLYGDVPFIE ERHRH RFEV NPNLIKQFEQ NDLSFVGQDV DGDRMEIIEL ANHPYFVGVQ FHPEFSSRPM KPSPPYLGLL LAATGNLNAY LQQGCK LSS SDRYSDASDD SFSEPRIAEL EIS

UniProtKB: CTP synthase 2

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Macromolecule #2: CYTIDINE-5'-TRIPHOSPHATE

MacromoleculeName: CYTIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 8 / Formula: CTP
Molecular weightTheoretical: 483.156 Da
Chemical component information

ChemComp-CTP:
CYTIDINE-5'-TRIPHOSPHATE

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 8 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.9
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 90.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 46236
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)

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