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- PDB-8f19: Cryo-EM structure of Kap114 bound to Gsp1 (RanGTP) -

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Basic information

Entry
Database: PDB / ID: 8f19
TitleCryo-EM structure of Kap114 bound to Gsp1 (RanGTP)
Components
  • GTP-binding nuclear protein GSP1/CNR1
  • Importin subunit beta-5
KeywordsPROTEIN TRANSPORT/NUCLEAR PROTEIN / Karyopherin Beta / Nuclear Transport / GTPase / PROTEIN TRANSPORT-NUCLEAR PROTEIN complex
Function / homology
Function and homology information


regulation of cell cycle phase transition / Postmitotic nuclear pore complex (NPC) reformation / exonucleolytic trimming to generate mature 3'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / regulation of nucleocytoplasmic transport / poly(A)+ mRNA export from nucleus / nuclear import signal receptor activity / NLS-bearing protein import into nucleus / nucleus organization / mRNA transport / ribosomal subunit export from nucleus ...regulation of cell cycle phase transition / Postmitotic nuclear pore complex (NPC) reformation / exonucleolytic trimming to generate mature 3'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / regulation of nucleocytoplasmic transport / poly(A)+ mRNA export from nucleus / nuclear import signal receptor activity / NLS-bearing protein import into nucleus / nucleus organization / mRNA transport / ribosomal subunit export from nucleus / nuclear pore / small GTPase binding / protein import into nucleus / nuclear envelope / GTPase activity / GTP binding / nucleus / cytosol / cytoplasm
Similarity search - Function
small GTPase Ran family profile. / Ran GTPase / Importin-beta N-terminal domain profile. / Importin-beta N-terminal domain / Importin-beta N-terminal domain / Importin-beta, N-terminal domain / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase ...small GTPase Ran family profile. / Ran GTPase / Importin-beta N-terminal domain profile. / Importin-beta N-terminal domain / Importin-beta N-terminal domain / Importin-beta, N-terminal domain / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Armadillo-like helical / Small GTP-binding protein domain / Armadillo-type fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / GTP-binding nuclear protein GSP1/CNR1 / Importin subunit beta-5
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.49 Å
AuthorsJiou, J. / Chook, Y.M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM141461-02 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Mechanism of RanGTP priming H2A-H2B release from Kap114 in an atypical RanGTP•Kap114•H2A-H2B complex.
Authors: Jenny Jiou / Joy M Shaffer / Natalia E Bernades / Ho Yee Joyce Fung / Juliana Kikumoto Dias / Sheena D'Arcy / Yuh Min Chook /
Abstract: Previously, we showed that the nuclear import receptor Importin-9 wraps around the H2A-H2B core to chaperone and transport it from the cytoplasm to the nucleus. However, unlike most nuclear import ...Previously, we showed that the nuclear import receptor Importin-9 wraps around the H2A-H2B core to chaperone and transport it from the cytoplasm to the nucleus. However, unlike most nuclear import systems where RanGTP dissociates cargoes from their importins, RanGTP binds stably to the Importin-9•H2A-H2B complex, and formation of the ternary RanGTP•Importin-9•H2A-H2B complex facilitates H2A-H2B release to the assembling nucleosome. It was unclear how RanGTP and the cargo H2A-H2B can bind simultaneously to an importin, and how interactions of the three components position H2A-H2B for release. Here, we show cryo-EM structures of Importin-9•RanGTP and of its yeast homolog Kap114, including Kap114•RanGTP, Kap114•H2A-H2B, and RanGTP•Kap114•H2A-H2B, to explain how the conserved Kap114 binds H2A-H2B and RanGTP simultaneously and how the GTPase primes histone transfer to the nucleosome. In the ternary complex, RanGTP binds to the N-terminal repeats of Kap114 in the same manner as in the Kap114/Importin-9•RanGTP complex, and H2A-H2B binds via its acidic patch to the Kap114 C-terminal repeats much like in the Kap114/Importin-9•H2A-H2B complex. Ran binds to a different conformation of Kap114 in the ternary RanGTP•Kap114•H2A-H2B complex. Here, Kap114 no longer contacts the H2A-H2B surface proximal to the H2A docking domain that drives nucleosome assembly, positioning it for transfer to the assembling nucleosome or to dedicated H2A-H2B chaperones in the nucleus.
History
DepositionNov 4, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 26, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Assembly

Deposited unit
A: Importin subunit beta-5
B: GTP-binding nuclear protein GSP1/CNR1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)134,9854
Polymers134,4372
Non-polymers5472
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: equilibrium centrifugation
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Importin subunit beta-5 / 114 kDa karyopherin / Karyopherin subunit beta-5 / Karyopherin-114


Mass: 114019.695 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: KAP114, YGL241W, HRC1004
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P53067
#2: Protein GTP-binding nuclear protein GSP1/CNR1 / Chromosome stability protein 17 / GTPase Ran homolog / Genetic suppressor of PRP20-1


Mass: 20417.590 Da / Num. of mol.: 1 / Mutation: Q71L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: GSP1, CNR1, CST17, YLR293C, L8003.19
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P32835
#3: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: Complex of Kap114 bound to Gsp1 (RanGTP) / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 7.5
Details: 20 mM Tris pH 7.5, 300 mM NaCl, 2 mM MgCl2, 1 mM TCEP, and 0.1% NP-40
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisaminomethane(HOCH2)3CNH21
2300 mMSodium ChlorideNaCl1
32 mMMagnesium ChlorideMgCl21
41 mMTris(2-carboxyethyl)phosphine hydrochlorideC9H15O6P1
50.1 %Nonyl phenoxypolyethoxylethanolH(C2H4O)nO(C6H4)C9H191
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 1 Kap114 to 1.2 Gsp1 (RanGTP) ratio
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 4 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: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 277305 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038860
ELECTRON MICROSCOPYf_angle_d0.5712012
ELECTRON MICROSCOPYf_dihedral_angle_d6.5971161
ELECTRON MICROSCOPYf_chiral_restr0.041429
ELECTRON MICROSCOPYf_plane_restr0.0041512

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