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- PDB-7tu7: Structure of the L. blandensis dGTPase H125A mutant bound to dGTP -

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Basic information

Entry
Database: PDB / ID: 7tu7
TitleStructure of the L. blandensis dGTPase H125A mutant bound to dGTP
ComponentsdGTP triphosphohydrolaseDGTPase
KeywordsHYDROLASE / dGTPase / nucleotide binding
Function / homology
Function and homology information


triphosphoric monoester hydrolase activity
Similarity search - Function
Deoxyguanosinetriphosphate triphosphohydrolase, C-terminal / Deoxyguanosinetriphosphate triphosphohydrolase, central domain superfamily / dNTP triphosphohydrolase / HD domain profile. / HD domain / HD domain / Metal dependent phosphohydrolases with conserved 'HD' motif. / HD/PDEase domain
Similarity search - Domain/homology
2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE / dGTP triphosphohydrolase
Similarity search - Component
Biological speciesLeeuwenhoekiella blandensis MED217 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsKlemm, B.P. / Sikkema, A.P. / Hsu, A.L. / Borgnia, M.J. / Schaaper, R.M.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZIAES050165 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZICES103326 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZIAES101905 United States
CitationJournal: J Biol Chem / Year: 2022
Title: High-resolution structures of the SAMHD1 dGTPase homolog from Leeuwenhoekiella blandensis reveal a novel mechanism of allosteric activation by dATP.
Authors: Bradley P Klemm / Andrew P Sikkema / Allen L Hsu / James C Horng / Traci M Tanaka Hall / Mario J Borgnia / Roel M Schaaper /
Abstract: Deoxynucleoside triphosphate (dNTP) triphosphohydrolases (dNTPases) are important enzymes that may perform multiple functions in the cell, including regulating the dNTP pools and contributing to ...Deoxynucleoside triphosphate (dNTP) triphosphohydrolases (dNTPases) are important enzymes that may perform multiple functions in the cell, including regulating the dNTP pools and contributing to innate immunity against viruses. Among the homologs that are best studied are human sterile alpha motif and HD domain-containing protein 1 (SAMHD1), a tetrameric dNTPase, and the hexameric Escherichia coli dGTPase; however, it is unclear whether these are representative of all dNTPases given their wide distribution throughout life. Here, we investigated a hexameric homolog from the marine bacterium Leeuwenhoekiella blandensis, revealing that it is a dGTPase that is subject to allosteric activation by dATP, specifically. Allosteric regulation mediated solely by dATP represents a novel regulatory feature among dNTPases that may facilitate maintenance of cellular dNTP pools in L. blandensis. We present high-resolution X-ray crystallographic structures (1.80-2.26 Å) in catalytically important conformations as well as cryo-EM structures (2.1-2.7 Å) of the enzyme bound to dGTP and dATP ligands. The structures, the highest resolution cryo-EM structures of any SAMHD1-like dNTPase to date, reveal an intact metal-binding site with the dGTP substrate coordinated to three metal ions. These structural and biochemical data yield insights into the catalytic mechanism and support a conserved catalytic mechanism for the tetrameric and hexameric dNTPase homologs. We conclude that the allosteric activation by dATP appears to rely on structural connectivity between the allosteric and active sites, as opposed to the changes in oligomeric state upon ligand binding used by SAMHD1.
History
DepositionFeb 2, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 1, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 15, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Jul 20, 2022Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.3Feb 21, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: dGTP triphosphohydrolase
B: dGTP triphosphohydrolase
C: dGTP triphosphohydrolase
D: dGTP triphosphohydrolase
E: dGTP triphosphohydrolase
F: dGTP triphosphohydrolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)319,41830
Polymers315,9376
Non-polymers3,48124
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, Observed particles appear to be hexameric, gel filtration, Runs as a single peak, approximately homohexamer in size
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
dGTP triphosphohydrolase / DGTPase


Mass: 52656.176 Da / Num. of mol.: 6 / Mutation: H125A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Leeuwenhoekiella blandensis MED217 (bacteria)
Strain: CECT 7118 / CCUG 51940 / MED217 / Gene: MED217_16760 / Plasmid: pMCSG7 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A3XHN1, dGTPase
#2: Chemical
ChemComp-DGT / 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE / Deoxyguanosine triphosphate


Mass: 507.181 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: dGTP triphosphohydrolaseDGTPase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Leeuwenhoekiella blandensis MED217 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pMCSG7
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris1
2100 mMsodium chlorideNaClSodium chloride1
315 mMmagnesium chlorideMgCl21
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Hexamer concentration is listed. 10 mM dGTP was added to the peak fraction after gel filtration.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 290 K / Details: 2.5 second blot time (front)

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 45000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 8.4 sec. / Electron dose: 54 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 391
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 60

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Processing

EM software
IDNameVersionCategory
1RELIONparticle selection
4CTFFIND4.1CTF correction
7PHENIXmodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
13PHENIXmodel refinement
Image processingDetails: 367 micrographs used after manually eliminating micrographs with poor CTF fit.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 256855 / Details: Laplacian-of-Gaussian auto-picking
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 120582 / Algorithm: FOURIER SPACE / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: Apo Cryo-EM model was fit into the EM map for building. dGTP ligands were initially built into the density using Coot.

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