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- PDB-8puu: Giardia intestinalis deoxyadenosine kinase forms a functional tetramer -

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Basic information

Entry
Database: PDB / ID: 8puu
TitleGiardia intestinalis deoxyadenosine kinase forms a functional tetramer
Components(Deoxynucleoside kinase) x 2
KeywordsTRANSFERASE / deoxyadenosine kinase / homotetramer / Giardia intestinales
Function / homology
Function and homology information


deoxynucleoside kinase activity / ATP binding / cytoplasm
Similarity search - Function
Deoxynucleoside kinase / : / Deoxynucleoside kinase domain / Deoxynucleoside kinase / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
2'-DEOXYADENOSINE-5'-MONOPHOSPHATE / 2'-DEOXYADENOSINE-5'-DIPHOSPHATE / Deoxynucleoside kinase
Similarity search - Component
Biological speciesGiardia intestinalis (eukaryote)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 Å
AuthorsRanjbarian, F. / Rafie, K. / Shankar, K. / Krakovka, S. / Svaerd, S. / Carlson, L.-A. / Hofer, A.
Funding support Sweden, 4items
OrganizationGrant numberCountry
Swedish Research Council2018-05851 Sweden
Swedish Research Council2021-01145 Sweden
Knut and Alice Wallenberg Foundation Sweden
Swedish Research Council2022-00593 Sweden
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Tetramerization of deoxyadenosine kinase meets the demands of a DNA replication substrate challenge in Giardia intestinalis.
Authors: Farahnaz Ranjbarian / Karim Rafie / Kasturika Shankar / Sascha Krakovka / Staffan G Svärd / Lars-Anders Carlson / Anders Hofer /
Abstract: The protozoan parasite Giardia intestinalis is one of only a few organisms lacking de novo synthesis of DNA building blocks (deoxyribonucleotides). Instead, the parasite relies exclusively on ...The protozoan parasite Giardia intestinalis is one of only a few organisms lacking de novo synthesis of DNA building blocks (deoxyribonucleotides). Instead, the parasite relies exclusively on salvaging deoxyadenosine and other deoxyribonucleosides from its host environment. Here, we report that G. intestinalis has a deoxyribonucleoside kinase with a 1000-fold higher catalytic efficiency (kcat/KM) for deoxyadenosine than the corresponding mammalian kinases and can thereby provide sufficient deoxyadenosine triphosphate levels for DNA synthesis despite the lack of de novo synthesis. Several deoxyadenosine analogs were also potent substrates and showed comparable EC50 values on cultured G. intestinalis cells as metronidazole, the current first-line treatment, with the additional advantage of being effective against metronidazole-resistant parasites. Structural analysis using cryo-EM and X-ray crystallography showed that the enzyme is unique within its family of deoxyribonucleoside kinases by forming a tetramer stabilized by extended N- and C-termini in a novel dimer-dimer interaction. Removal of the two termini resulted in lost ability to form tetramers and a markedly reduced affinity for the deoxyribonucleoside substrate. The development of highly efficient deoxyribonucleoside kinases via oligomerization may represent a critical evolutionary adaptation in organisms that rely solely on deoxyribonucleoside salvage.
History
DepositionJul 17, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 30, 2024Provider: repository / Type: Initial release
Revision 2.0Dec 18, 2024Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Other / Polymer sequence / Source and taxonomy / Structure summary
Category: atom_site / cell ...atom_site / cell / citation / citation_author / entity / entity_name_com / entity_poly / entity_poly_seq / entity_src_gen / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_poly_seq_scheme / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_residues / struct / struct_asym / struct_conf / struct_ref / struct_ref_seq / struct_sheet_range
Item: _cell.Z_PDB / _citation.country ..._cell.Z_PDB / _citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _pdbx_entity_nonpoly.entity_id / _pdbx_nonpoly_scheme.entity_id / _pdbx_struct_sheet_hbond.range_1_label_seq_id / _pdbx_struct_sheet_hbond.range_2_label_seq_id / _struct.title / _struct_asym.entity_id / _struct_conf.beg_label_seq_id / _struct_conf.end_label_seq_id / _struct_ref_seq.db_align_beg / _struct_ref_seq.db_align_end / _struct_ref_seq.pdbx_auth_seq_align_beg / _struct_ref_seq.pdbx_auth_seq_align_end / _struct_ref_seq.ref_id / _struct_ref_seq.seq_align_end / _struct_sheet_range.beg_label_seq_id / _struct_sheet_range.end_label_seq_id
Revision 2.1Jan 1, 2025Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Deoxynucleoside kinase
B: Deoxynucleoside kinase
C: Deoxynucleoside kinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)58,3145
Polymers57,5723
Non-polymers7422
Water2,000111
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, cryo-electron microscopy experiments have been performed to validate the tetramer formation in solution., assay for oligomerization, Mass-photometry experiments were ...Evidence: electron microscopy, cryo-electron microscopy experiments have been performed to validate the tetramer formation in solution., assay for oligomerization, Mass-photometry experiments were performed to validate the tetramerization of the protein.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)103.130, 103.130, 147.109
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number154
Space group name H-MP3221
Symmetry operation#1: x,y,z
#2: -y,x-y,z+2/3
#3: -x+y,-x,z+1/3
#4: x-y,-y,-z+1/3
#5: -x,-x+y,-z+2/3
#6: y,x,-z

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Components

#1: Protein Deoxynucleoside kinase / dAK


Mass: 28436.518 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Giardia intestinalis (eukaryote) / Gene: GL50803_0017451 / Production host: Escherichia coli (E. coli) / References: UniProt: A8B9V6, 2'-deoxyadenosine kinase
#2: Protein/peptide Deoxynucleoside kinase / dAK


Mass: 698.854 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Giardia intestinalis (eukaryote) / Production host: Escherichia coli (E. coli) / References: 2'-deoxyadenosine kinase
#3: Chemical ChemComp-DAT / 2'-DEOXYADENOSINE-5'-DIPHOSPHATE / DADP


Mass: 411.202 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O9P2 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-D5M / 2'-DEOXYADENOSINE-5'-MONOPHOSPHATE


Mass: 331.222 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H14N5O6P / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 111 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 4 Å3/Da / Density % sol: 69.3 % / Description: Cube shaped crystals
Crystal growTemperature: 294.15 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: Morpheus Screen condition H3: 0.1 M amino acids, 0.1 M Buffer 1 pH 6.5, 30% GOL_P4K
Temp details: Temperature controlled room

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: cryo-stream / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: MAX IV / Beamline: BioMAX / Wavelength: 0.976254 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Oct 23, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.976254 Å / Relative weight: 1
ReflectionResolution: 2.1→49.04 Å / Num. obs: 53424 / % possible obs: 99.72 % / Redundancy: 20 % / Biso Wilson estimate: 50.25 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.092 / Rpim(I) all: 0.03 / Rrim(I) all: 0.097 / Net I/σ(I): 15.4
Reflection shellResolution: 2.1→2.175 Å / Mean I/σ(I) obs: 2.6 / Num. unique obs: 5191 / CC1/2: 0.802 / % possible all: 99.12

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Processing

Software
NameVersionClassification
PHENIX(1.20.1_4487: ???)refinement
MxCuBEdata collection
Coot0.8.9.2.ELmodel building
PHASER2.8.3phasing
XDSVERSION Jan 10, 2022 BUILT=20220820data reduction
Aimless0.7.9data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.1→49.04 Å / Cross valid method: FREE R-VALUE
RfactorNum. reflection% reflection
Rfree0.2247 -5 %
Rwork0.2 --
obs-53246 99.72 %
Displacement parametersBiso mean: 54.22 Å2
Refinement stepCycle: LAST / Resolution: 2.1→49.04 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3651 0 26 111 3788

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