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- PDB-8sgh: Cryo-EM structure of Karyopherin-beta2 bound to HNRNPH2 PY-NLS -

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Basic information

Entry
Database: PDB / ID: 8sgh
TitleCryo-EM structure of Karyopherin-beta2 bound to HNRNPH2 PY-NLS
Components
  • Heterogeneous nuclear ribonucleoprotein H2, N-terminally processed
  • Transportin-1
KeywordsTRANSPORT PROTEIN / RNA-binding protein TNPO1 Cryo-EM KapB2 HNRNP
Function / homology
Function and homology information


Tristetraprolin (TTP, ZFP36) binds and destabilizes mRNA / Intraflagellar transport / Postmitotic nuclear pore complex (NPC) reformation / nuclear import signal receptor activity / nuclear localization sequence binding / regulation of RNA splicing / Processing of Capped Intron-Containing Pre-mRNA / mRNA Splicing - Major Pathway / cilium / small GTPase binding ...Tristetraprolin (TTP, ZFP36) binds and destabilizes mRNA / Intraflagellar transport / Postmitotic nuclear pore complex (NPC) reformation / nuclear import signal receptor activity / nuclear localization sequence binding / regulation of RNA splicing / Processing of Capped Intron-Containing Pre-mRNA / mRNA Splicing - Major Pathway / cilium / small GTPase binding / protein import into nucleus / postsynaptic density / ribonucleoprotein complex / RNA binding / extracellular exosome / nucleoplasm / membrane / nucleus / cytosol / cytoplasm
Similarity search - Function
Zinc finger, CHHC-type / RNPHF zinc finger / : / Importin beta family / HEAT-like repeat / HEAT repeat / HEAT repeat / Importin-beta N-terminal domain profile. / Importin-beta N-terminal domain / Importin-beta N-terminal domain ...Zinc finger, CHHC-type / RNPHF zinc finger / : / Importin beta family / HEAT-like repeat / HEAT repeat / HEAT repeat / Importin-beta N-terminal domain profile. / Importin-beta N-terminal domain / Importin-beta N-terminal domain / Importin-beta, N-terminal domain / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / RNA-binding domain superfamily / Armadillo-like helical / Armadillo-type fold / Nucleotide-binding alpha-beta plait domain superfamily
Similarity search - Domain/homology
Heterogeneous nuclear ribonucleoprotein H2 / Transportin-1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.17 Å
AuthorsGonzalez, A. / Fung, H.Y.J. / Chook, Y.M.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM141461 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM069909 United States
Welch FoundationI-1532 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS120465 United States
CitationJournal: Structure / Year: 2023
Title: A new Karyopherin-β2 binding PY-NLS epitope of HNRNPH2 linked to neurodevelopmental disorders.
Authors: Abner Gonzalez / Hong Joo Kim / Brian D Freibaum / Ho Yee Joyce Fung / Chad A Brautigam / J Paul Taylor / Yuh Min Chook /
Abstract: The HNRNPH2 proline-tyrosine nuclear localization signal (PY-NLS) is mutated in HNRNPH2-related X-linked neurodevelopmental disorder, causing the normally nuclear HNRNPH2 to accumulate in the ...The HNRNPH2 proline-tyrosine nuclear localization signal (PY-NLS) is mutated in HNRNPH2-related X-linked neurodevelopmental disorder, causing the normally nuclear HNRNPH2 to accumulate in the cytoplasm. We solved the cryoelectron microscopy (cryo-EM) structure of Karyopherin-β2/Transportin-1 bound to the HNRNPH2 PY-NLS to understand importin-NLS recognition and disruption in disease. HNRNPH2 RPGPY is a typical R-X-P-Y motif comprising PY-NLS epitopes 2 and 3, followed by an additional Karyopherin-β2-binding epitope, we term epitope 4, at residues DRP; no density is present for PY-NLS epitope 1. Disease variant mutations at epitopes 2-4 impair Karyopherin-β2 binding and cause aberrant cytoplasmic accumulation in cells, emphasizing the role of nuclear import defect in disease. Sequence/structure analysis suggests that strong PY-NLS epitopes 4 are rare and thus far limited to close paralogs of HNRNPH2, HNRNPH1, and HNRNPF. Epitope 4-binidng hotspot Karyopherin-β2 W373 corresponds to close paralog Karyopherin-β2b/Transportin-2 W370, a pathological variant site in neurodevelopmental abnormalities, suggesting that Karyopherin-β2b/Transportin-2-HNRNPH2/H1/F interactions may be compromised in the abnormalities.
History
DepositionApr 12, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 14, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 21, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Aug 16, 2023Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Transportin-1
B: Heterogeneous nuclear ribonucleoprotein H2, N-terminally processed


Theoretical massNumber of molelcules
Total (without water)115,7332
Polymers115,7332
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Transportin-1 / Importin beta-2 / Karyopherin beta-2 / M9 region interaction protein / MIP


Mass: 101738.812 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TNPO1, KPNB2, MIP1, TRN / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q92973
#2: Protein Heterogeneous nuclear ribonucleoprotein H2, N-terminally processed


Mass: 13993.811 Da / Num. of mol.: 1 / Fragment: UNP residues 103-225
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HNRNPH2, FTP3, HNRPH2 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P55795

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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: Karyopherin-beta2 bound to hnRNP H2 PY-NLS / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Details: 20 mM Tris-HCl pH 7.5, 150 nM NaCl, 2 mM BME, 0.003125% [w/v] NP-40
SpecimenConc.: 2.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K

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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: 2200 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameCategory
1cryoSPARCparticle selection
4cryoSPARCCTF correction
7ISOLDEmodel fitting
8UCSF ChimeraXmodel fitting
9Cootmodel fitting
11cryoSPARCinitial Euler assignment
12cryoSPARCfinal Euler assignment
13cryoSPARCclassification
14cryoSPARC3D reconstruction
15PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 208572 / Details: Non-uniform refinement / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingDetails: Initial docking performed using Chimera, then manual model building by Coot, ISOLDE in ChimerX and Phenix real-space refinement.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00313814
ELECTRON MICROSCOPYf_angle_d0.66625090
ELECTRON MICROSCOPYf_dihedral_angle_d9.1692173
ELECTRON MICROSCOPYf_chiral_restr0.0411083
ELECTRON MICROSCOPYf_plane_restr0.0092026

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