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- PDB-9kjt: The cryo-EM structure of human PNPase in the closed conformation -

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Basic information

Entry
Database: PDB / ID: 9kjt
TitleThe cryo-EM structure of human PNPase in the closed conformation
ComponentsPolyribonucleotide nucleotidyltransferase 1, mitochondrial
KeywordsRNA BINDING PROTEIN / 3'-to-5' exoribonuclease / RNA degradation / RNA import / mitochondria
Function / homology
Function and homology information


RNA import into mitochondrion / mitochondrial mRNA polyadenylation / mitochondrial degradosome / mitochondrial mRNA catabolic process / positive regulation of mitochondrial RNA catabolic process / mitochondrial RNA 3'-end processing / Mitochondrial RNA degradation / positive regulation of miRNA catabolic process / mitochondrial RNA 5'-end processing / poly(G) binding ...RNA import into mitochondrion / mitochondrial mRNA polyadenylation / mitochondrial degradosome / mitochondrial mRNA catabolic process / positive regulation of mitochondrial RNA catabolic process / mitochondrial RNA 3'-end processing / Mitochondrial RNA degradation / positive regulation of miRNA catabolic process / mitochondrial RNA 5'-end processing / poly(G) binding / polyribonucleotide nucleotidyltransferase / polyribonucleotide nucleotidyltransferase activity / mitochondrial RNA catabolic process / nuclear polyadenylation-dependent mRNA catabolic process / positive regulation of mRNA catabolic process / regulation of cellular senescence / rRNA import into mitochondrion / regulation of cellular respiration / response to growth hormone / RNA catabolic process / miRNA binding / poly(U) RNA binding / protein homotrimerization / mRNA catabolic process / response to cAMP / cellular response to interferon-beta / liver regeneration / mitochondrion organization / protein homooligomerization / mitochondrial intermembrane space / mRNA processing / cellular response to oxidative stress / 3'-5'-RNA exonuclease activity / ribosome / mitochondrial matrix / endoplasmic reticulum membrane / mitochondrion / RNA binding / identical protein binding / cytoplasm / 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
Polyribonucleotide nucleotidyltransferase 1, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.84 Å
AuthorsLi, Y.C. / Yuan, H.S.
Funding support Taiwan, 1items
OrganizationGrant numberCountry
Academia Sinica (Taiwan) Taiwan
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Structural insights into human PNPase in health and disease.
Authors: Yi-Ching Li / Chun-Hsiung Wang / Malay Patra / Yi-Ping Chen / Wei-Zen Yang / Hanna S Yuan /
Abstract: Human polynucleotide phosphorylase (hPNPase) is a 3'-to-5' exoribonuclease located in mitochondria, where it plays crucial roles in RNA degradation and RNA import. Mutations in hPNPase can impair ...Human polynucleotide phosphorylase (hPNPase) is a 3'-to-5' exoribonuclease located in mitochondria, where it plays crucial roles in RNA degradation and RNA import. Mutations in hPNPase can impair these functions, leading to various mitochondrial dysfunctions and diseases. However, the mechanisms by which hPNPase switches between its roles as an RNA-degrading enzyme and an RNA carrier, as well as how disease-associated mutations may affect these distinct functions, remain unclear. In this study, we present cryo-electron microscopy structures of hPNPase, highlighting the flexibility of its S1 domains, which cap the ring-like RNA-degradation chamber and shift between two distinctive open and closed conformations. We further demonstrate by small-angle X-ray scattering and biochemical analyses that the disease-associated mutations P467S and G499R impair hPNPase's stem-loop RNA-binding and degradation activities by limiting the S1 domain's ability to transition from an open to closed state. Conversely, the D713Y mutation, located within the S1 domain, does not affect the RNA-binding affinity of hPNPase, but diminishes its interaction with Suv3 helicase for cooperative degradation of structured RNA. Collectively, these findings underscore the critical role of S1 domain mobility in capturing structured RNA for degradation and import, as well as its involvement in mitochondrial degradosome assembly. Our study thereby reveals the molecular mechanism of hPNPase in RNA binding and degradation, and the multiple molecular defects that could be induced by disease-linked mutations in hPNPase.
History
DepositionNov 12, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 12, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Revision 1.1Jul 2, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1Jul 2, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Polyribonucleotide nucleotidyltransferase 1, mitochondrial
B: Polyribonucleotide nucleotidyltransferase 1, mitochondrial
C: Polyribonucleotide nucleotidyltransferase 1, mitochondrial


Theoretical massNumber of molelcules
Total (without water)247,2753
Polymers247,2753
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Polyribonucleotide nucleotidyltransferase 1, mitochondrial / 3'-5' RNA exonuclease OLD35 / PNPase old-35 / Polynucleotide phosphorylase 1 / PNPase 1 / ...3'-5' RNA exonuclease OLD35 / PNPase old-35 / Polynucleotide phosphorylase 1 / PNPase 1 / Polynucleotide phosphorylase-like protein


Mass: 82425.031 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PNPT1, PNPASE / Production host: Escherichia coli (E. coli)
References: UniProt: Q8TCS8, polyribonucleotide nucleotidyltransferase
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: Human PNPase in open form / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1600 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 809000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00616680
ELECTRON MICROSCOPYf_angle_d0.62922578
ELECTRON MICROSCOPYf_dihedral_angle_d3.7942295
ELECTRON MICROSCOPYf_chiral_restr0.0422634
ELECTRON MICROSCOPYf_plane_restr0.0032934

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