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- EMDB-40455: Cryo-EM structure of Karyopherin-beta2 bound to HNRNPH2 PY-NLS -

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Basic information

Entry
Database: EMDB / ID: EMD-40455
TitleCryo-EM structure of Karyopherin-beta2 bound to HNRNPH2 PY-NLS
Map data
Sample
  • Complex: Karyopherin-beta2 bound to hnRNP H2 PY-NLS
    • Protein or peptide: Transportin-1
    • Protein or peptide: Heterogeneous nuclear ribonucleoprotein H2, N-terminally processed
KeywordsRNA-binding protein TNPO1 Cryo-EM KapB2 HNRNP / TRANSPORT PROTEIN
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 repeat / HEAT repeat / HEAT-like repeat / Importin-beta N-terminal domain profile. / Importin-beta N-terminal domain / Importin-beta N-terminal domain / Importin-beta, N-terminal domain ...Zinc finger, CHHC-type / RNPHF zinc finger / Importin beta family / HEAT repeat / HEAT repeat / HEAT-like 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.17 Å
AuthorsGonzalez A / Fung HYJ / Chook YM
Funding support United States, 4 items
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, 2023-
Header (metadata) releaseJun 14, 2023-
Map releaseJun 14, 2023-
UpdateAug 16, 2023-
Current statusAug 16, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40455.png

Downloads & links

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Map

FileDownload / File: emd_40455.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.415 Å
Density
Contour LevelBy AUTHOR: 0.021
Minimum - Maximum-0.04081724 - 0.12058783
Average (Standard dev.)0.00033213876 (±0.0030574289)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 249.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_40455_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_40455_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Karyopherin-beta2 bound to hnRNP H2 PY-NLS

EntireName: Karyopherin-beta2 bound to hnRNP H2 PY-NLS
Components
  • Complex: Karyopherin-beta2 bound to hnRNP H2 PY-NLS
    • Protein or peptide: Transportin-1
    • Protein or peptide: Heterogeneous nuclear ribonucleoprotein H2, N-terminally processed

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Supramolecule #1: Karyopherin-beta2 bound to hnRNP H2 PY-NLS

SupramoleculeName: Karyopherin-beta2 bound to hnRNP H2 PY-NLS / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Transportin-1

MacromoleculeName: Transportin-1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 101.738812 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GGSKMEYEWK PDEQGLQQIL QLLKESQSPD TTIQRTVQQK LEQLNQYPDF NNYLIFVLTK LKSEDEPTRS LSGLILKNNV KAHFQNFPN GVTDFIKSEC LNNIGDSSPL IRATVGILIT TIASKGELQN WPDLLPKLCS LLDSEDYNTC EGAFGALQKI C EDSAEILD ...String:
GGSKMEYEWK PDEQGLQQIL QLLKESQSPD TTIQRTVQQK LEQLNQYPDF NNYLIFVLTK LKSEDEPTRS LSGLILKNNV KAHFQNFPN GVTDFIKSEC LNNIGDSSPL IRATVGILIT TIASKGELQN WPDLLPKLCS LLDSEDYNTC EGAFGALQKI C EDSAEILD SDVLDRPLNI MIPKFLQFFK HSSPKIRSHA VACVNQFIIS RTQALMLHID SFIENLFALA GDEEPEVRKN VC RALVMLL EVRMDRLLPH MHNIVEYMLQ RTQDQDENVA LEACEFWLTL AEQPICKDVL VRHLPKLIPV LVNGMKYSDI DII LLKGDV EEDETIPDSE QDIRPRFHRS RTVAQQHDED GIEEEDDDDD EIDDDDTISD WNLRKCSAAA LDVLANVYRD ELLP HILPL LKELLFHHEW VVKESGILVL GAIAEGCMQG MIPYLPELIP HLIQCLSDKK ALVRSITCWT LSRYAHWVVS QPPDT YLKP LMTELLKRIL DSNKRVQEAA CSAFATLEEE ACTELVPYLA YILDTLVFAF SKYQHKNLLI LYDAIGTLAD SVGHHL NKP EYIQMLMPPL IQKWNMLKDE DKDLFPLLEC LSSVATALQS GFLPYCEPVY QRCVNLVQKT LAQAMLNNAQ PDQYEAP DK DFMIVALDLL SGLAEGLGGN IEQLVARSNI LTLMYQCMQD KMPEVRQSSF ALLGDLTKAC FQHVKPCIAD FMPILGTN L NPEFISVCNN ATWAIGEISI QMGIEMQPYI PMVLHQLVEI INRPNTPKTL LENTAITIGR LGYVCPQEVA PMLQQFIRP WCTSLRNIRD NEEKDSAFRG ICTMISVNPS GVIQDFIFFC DAVASWINPK DDLRDMFCKI LHGFKNQVGD ENWRRFSDQF PLPLKERLA AFYGV

UniProtKB: Transportin-1

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Macromolecule #2: Heterogeneous nuclear ribonucleoprotein H2, N-terminally processed

MacromoleculeName: Heterogeneous nuclear ribonucleoprotein H2, N-terminally processed
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.993811 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GGSNSPDTAN DGFVRLRGLP FGCSKEEIVQ FFSGLEIVPN GMTLPVDFQG RSTGEAFVQF ASQEIAEKAL KKHKERIGHR YIEIFKSSR AEVRTHYDPP RKLMAMQRPG PYDRPGAGRG YNSIGRG

UniProtKB: Heterogeneous nuclear ribonucleoprotein H2

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.7 mg/mL
BufferpH: 7.5
Details: 20 mM Tris-HCl pH 7.5, 150 nM NaCl, 2 mM BME, 0.003125% [w/v] NP-40
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 7 / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Details: Non-uniform refinement / Number images used: 208572

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Atomic model buiding 1

DetailsInitial docking performed using Chimera, then manual model building by Coot, ISOLDE in ChimerX and Phenix real-space refinement.
Output model

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

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