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- PDB-8t7t: CryoEM structure of the HisRS-like domain of human GCN2 -

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Basic information

Entry
Database: PDB / ID: 8t7t
TitleCryoEM structure of the HisRS-like domain of human GCN2
ComponentseIF-2-alpha kinase GCN2
KeywordsTRANSFERASE / GCN2 / HisRS / pseudoenzyme / translation
Function / homology
Function and homology information


regulation of translational initiation by eIF2 alpha phosphorylation / positive regulation of translational initiation in response to starvation / : / GCN2-mediated signaling / eukaryotic translation initiation factor 2alpha kinase activity / regulation of feeding behavior / negative regulation of translational initiation in response to stress / host-mediated suppression of viral genome replication / T cell activation involved in immune response / positive regulation of adaptive immune response ...regulation of translational initiation by eIF2 alpha phosphorylation / positive regulation of translational initiation in response to starvation / : / GCN2-mediated signaling / eukaryotic translation initiation factor 2alpha kinase activity / regulation of feeding behavior / negative regulation of translational initiation in response to stress / host-mediated suppression of viral genome replication / T cell activation involved in immune response / positive regulation of adaptive immune response / neuron projection extension / regulation of translational initiation / negative regulation of neuron differentiation / cellular response to cold / viral translation / Response of EIF2AK4 (GCN2) to amino acid deficiency / long-term memory / positive regulation of defense response to virus by host / negative regulation of translational initiation / cytosolic ribosome / cellular response to amino acid starvation / DNA damage checkpoint signaling / positive regulation of long-term synaptic potentiation / learning / cellular response to UV / protein autophosphorylation / defense response to virus / adaptive immune response / tRNA binding / protein phosphorylation / non-specific serine/threonine protein kinase / protein serine kinase activity / protein serine/threonine kinase activity / ATP binding / nucleus / cytoplasm / cytosol
Similarity search - Function
eIF-2-alpha kinase Gcn2 / Histidyl tRNA synthetase-related domain / Anticodon binding domain of tRNAs / RWD domain / RWD domain profile. / RWD / RWD domain / Class II Histidinyl-tRNA synthetase (HisRS)-like catalytic core domain / Histidyl-tRNA synthetase / : ...eIF-2-alpha kinase Gcn2 / Histidyl tRNA synthetase-related domain / Anticodon binding domain of tRNAs / RWD domain / RWD domain profile. / RWD / RWD domain / Class II Histidinyl-tRNA synthetase (HisRS)-like catalytic core domain / Histidyl-tRNA synthetase / : / Anticodon-binding domain superfamily / Ubiquitin-conjugating enzyme/RWD-like / Class II Aminoacyl-tRNA synthetase/Biotinyl protein ligase (BPL) and lipoyl protein ligase (LPL) / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
eIF-2-alpha kinase GCN2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsYin, J.Z. / Keszei, A.F.A. / Mazhab-Jafari, M.T. / Sicheri, F.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
CitationJournal: Structure / Year: 2024
Title: The HisRS-like domain of GCN2 is a pseudoenzyme that can bind uncharged tRNA.
Authors: Jay Z Yin / Alexander F A Keszei / Scott Houliston / Frantisek Filandr / Jonah Beenstock / Salima Daou / Julia Kitaygorodsky / David C Schriemer / Mohammad T Mazhab-Jafari / Anne-Claude ...Authors: Jay Z Yin / Alexander F A Keszei / Scott Houliston / Frantisek Filandr / Jonah Beenstock / Salima Daou / Julia Kitaygorodsky / David C Schriemer / Mohammad T Mazhab-Jafari / Anne-Claude Gingras / Frank Sicheri /
Abstract: GCN2 is a stress response kinase that phosphorylates the translation initiation factor eIF2α to inhibit general protein synthesis when activated by uncharged tRNA and stalled ribosomes. The presence ...GCN2 is a stress response kinase that phosphorylates the translation initiation factor eIF2α to inhibit general protein synthesis when activated by uncharged tRNA and stalled ribosomes. The presence of a HisRS-like domain in GCN2, normally associated with tRNA aminoacylation, led to the hypothesis that eIF2α kinase activity is regulated by the direct binding of this domain to uncharged tRNA. Here we solved the structure of the HisRS-like domain in the context of full-length GCN2 by cryoEM. Structure and function analysis shows the HisRS-like domain of GCN2 has lost histidine and ATP binding but retains tRNA binding abilities. Hydrogen deuterium exchange mass spectrometry, site-directed mutagenesis and computational docking experiments support a tRNA binding model that is partially shifted from that employed by bona fide HisRS enzymes. These results demonstrate that the HisRS-like domain of GCN2 is a pseudoenzyme and advance our understanding of GCN2 regulation and function.
History
DepositionJun 21, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 25, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: eIF-2-alpha kinase GCN2
B: eIF-2-alpha kinase GCN2


Theoretical massNumber of molelcules
Total (without water)374,4332
Polymers374,4332
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 eIF-2-alpha kinase GCN2 / Eukaryotic translation initiation factor 2-alpha kinase 4 / GCN2-like protein


Mass: 187216.578 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EIF2AK4, GCN2, KIAA1338 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q9P2K8, non-specific serine/threonine protein kinase

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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: dimeric HisRS-like domain of human GCN2 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
SpecimenEmbedding 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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
4cryoSPARCCTF correction
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 229225 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 78.49 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00197568
ELECTRON MICROSCOPYf_angle_d0.42110280
ELECTRON MICROSCOPYf_chiral_restr0.04041224
ELECTRON MICROSCOPYf_plane_restr0.00341306
ELECTRON MICROSCOPYf_dihedral_angle_d2.82011050

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