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- PDB-8vah: E.coli PNPase in complex with single 8-oxoG RNA -

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Basic information

Entry
Database: PDB / ID: 8vah
TitleE.coli PNPase in complex with single 8-oxoG RNA
Components
  • Polyribonucleotide nucleotidyltransferase
  • RNA (5'-R(P*AP*AP*AP*AP*AP*AP*A*A*A)-3')
  • RNA (5'-R(P*AP*CP*AP*(8GM))-3')
KeywordsRNA BINDING PROTEIN / PNPase / oxidized RNA / Phosphorolysis / 8-oxo G
Function / homology
Function and homology information


polyribonucleotide nucleotidyltransferase / polyribonucleotide nucleotidyltransferase activity / mRNA catabolic process / RNA processing / 3'-5'-RNA exonuclease activity / magnesium ion binding / RNA binding / cytosol
Similarity search - Function
Polyribonucleotide nucleotidyltransferase, RNA-binding domain superfamily / Polyribonucleotide nucleotidyltransferase / Polyribonucleotide nucleotidyltransferase, RNA-binding domain / Polyribonucleotide nucleotidyltransferase, RNA binding domain / Exoribonuclease, phosphorolytic domain 2 / 3' exoribonuclease family, domain 2 / Exoribonuclease, phosphorolytic domain 1 / PNPase/RNase PH domain superfamily / Exoribonuclease, PH domain 2 superfamily / 3' exoribonuclease family, domain 1 ...Polyribonucleotide nucleotidyltransferase, RNA-binding domain superfamily / Polyribonucleotide nucleotidyltransferase / Polyribonucleotide nucleotidyltransferase, RNA-binding domain / Polyribonucleotide nucleotidyltransferase, RNA binding domain / Exoribonuclease, phosphorolytic domain 2 / 3' exoribonuclease family, domain 2 / Exoribonuclease, phosphorolytic domain 1 / PNPase/RNase PH domain superfamily / Exoribonuclease, PH domain 2 superfamily / 3' exoribonuclease family, domain 1 / KH domain / K Homology domain, type 1 / Type-1 KH domain profile. / K Homology domain, type 1 superfamily / S1 domain profile. / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / K Homology domain / K homology RNA-binding domain / Ribosomal protein S5 domain 2-type fold / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
RNA / Polyribonucleotide nucleotidyltransferase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsKim, W. / Zhang, Y.J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM148356 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Selective 8-oxo-rG stalling occurs in the catalytic core of polynucleotide phosphorylase (PNPase) during degradation.
Authors: Lucas G Miller / Wantae Kim / Shawn Schowe / Kathleen Taylor / Runhua Han / Vashita Jain / Raeyeon Park / Mark Sherman / Janssen Fang / Haydee Ramirez / Andrew Ellington / Phanourios Tamamis ...Authors: Lucas G Miller / Wantae Kim / Shawn Schowe / Kathleen Taylor / Runhua Han / Vashita Jain / Raeyeon Park / Mark Sherman / Janssen Fang / Haydee Ramirez / Andrew Ellington / Phanourios Tamamis / Marino J E Resendiz / Y Jessie Zhang / Lydia Contreras /
Abstract: RNA oxidation, predominantly through the accumulation of 8-oxo-7,8-dihydroguanosine (8-oxo-rG), represents an important biomarker for cellular oxidative stress. Polynucleotide phosphorylase (PNPase) ...RNA oxidation, predominantly through the accumulation of 8-oxo-7,8-dihydroguanosine (8-oxo-rG), represents an important biomarker for cellular oxidative stress. Polynucleotide phosphorylase (PNPase) is a 3'-5' exoribonuclease that has been shown to preferentially recognize 8-oxo-rG-containing RNA and protect cells from oxidative stress. However, the impact of 8-oxo-rG on PNPase-mediated RNA degradation has not been studied. Here, we show that the presence of 8-oxo-rG in RNA leads to catalytic stalling of PNPase through in vitro RNA degradation experiments and electrophoretic analysis. We also link this stalling to the active site of the enzyme through resolution of single-particle cryo-EM structures for PNPase in complex with singly or doubly oxidized RNA oligonucleotides. Following identification of Arg399 as a key residue in recognition of both single and sequential 8-oxo-rG nucleotides, we perform follow-up in vitro analysis to confirm the importance of this residue in 8-oxo-rG-specific PNPase stalling. Finally, we investigate the effects of mutations to active site residues implicated in 8-oxo-rG binding through cell growth experiments under HO-induced oxidative stress. Specifically, Arg399 mutations show significant effects on cell growth under oxidative stress. Overall, we demonstrate that 8-oxo-rG-specific stalling of PNPase is relevant to bacterial survival under oxidative stress and speculate that this enzyme might associate with other cellular factors to mediate this stress.
History
DepositionDec 11, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 20, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Polyribonucleotide nucleotidyltransferase
B: Polyribonucleotide nucleotidyltransferase
C: Polyribonucleotide nucleotidyltransferase
G: RNA (5'-R(P*AP*AP*AP*AP*AP*AP*A*A*A)-3')
D: RNA (5'-R(P*AP*CP*AP*(8GM))-3')
E: RNA (5'-R(P*AP*CP*AP*(8GM))-3')
F: RNA (5'-R(P*AP*CP*AP*(8GM))-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)238,40010
Polymers238,3277
Non-polymers733
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Polyribonucleotide nucleotidyltransferase


Mass: 77189.844 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: pnp / Production host: Escherichia coli (E. coli) / References: UniProt: C4ZSQ5
#2: RNA chain RNA (5'-R(P*AP*AP*AP*AP*AP*AP*A*A*A)-3')


Mass: 2917.895 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#3: RNA chain RNA (5'-R(P*AP*CP*AP*(8GM))-3')


Mass: 1279.841 Da / Num. of mol.: 3 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestY
Has protein modificationN

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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: PNPase complexed with single 8-oxo G RNA / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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 / Nominal defocus max: 1900 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 69 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.18rc3_3805: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 705788 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00616272
ELECTRON MICROSCOPYf_angle_d0.61922035
ELECTRON MICROSCOPYf_dihedral_angle_d10.3242328
ELECTRON MICROSCOPYf_chiral_restr0.0462576
ELECTRON MICROSCOPYf_plane_restr0.0042883

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