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- PDB-7nzm: Cryo-EM structure of pre-dephosphorylation complex of phosphoryla... -

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Basic information

Entry
Database: PDB / ID: 7nzm
TitleCryo-EM structure of pre-dephosphorylation complex of phosphorylated eIF2alpha with trapped holophosphatase (PP1A_D64A/PPP1R15A/G-actin/DNase I)
Components
  • Actin, alpha skeletal muscle, intermediate form
  • Deoxyribonuclease-1
  • Eukaryotic translation initiation factor 2 subunit 1
  • Protein phosphatase 1 regulatory subunit 15A,Maltose/maltodextrin-binding periplasmic protein
  • Serine/threonine-protein phosphatase PP1-alpha catalytic subunit
KeywordsHYDROLASE / holophosphatase / PP1 / PPP1R15A / phosphorylated eIF2alpha
Function / homology
Function and homology information


fatty acid derivative binding / positive regulation of translational initiation in response to stress / positive regulation of endoplasmic reticulum stress-induced eIF2 alpha dephosphorylation / regulation of translational initiation by eIF2 alpha dephosphorylation / regulation of neutrophil mediated cytotoxicity / zymogen granule / regulation of acute inflammatory response / positive regulation of peptidyl-serine dephosphorylation / deoxyribonuclease I / regulation of translation in response to endoplasmic reticulum stress ...fatty acid derivative binding / positive regulation of translational initiation in response to stress / positive regulation of endoplasmic reticulum stress-induced eIF2 alpha dephosphorylation / regulation of translational initiation by eIF2 alpha dephosphorylation / regulation of neutrophil mediated cytotoxicity / zymogen granule / regulation of acute inflammatory response / positive regulation of peptidyl-serine dephosphorylation / deoxyribonuclease I / regulation of translation in response to endoplasmic reticulum stress / translation initiation ternary complex / protein phosphatase type 1 complex / glial limiting end-foot / response to kainic acid / Cellular response to mitochondrial stress / response to manganese-induced endoplasmic reticulum stress / positive regulation of type B pancreatic cell apoptotic process / negative regulation of translational initiation in response to stress / Response of EIF2AK1 (HRI) to heme deficiency / PTW/PP1 phosphatase complex / Recycling of eIF2:GDP / negative regulation of protein dephosphorylation / PERK-mediated unfolded protein response / PERK regulates gene expression / eukaryotic translation initiation factor 2 complex / peptidyl-serine dephosphorylation / regulation of translational initiation in response to stress / neutrophil activation involved in immune response / protein localization to endoplasmic reticulum / deoxyribonuclease I activity / protein phosphatase regulator activity / protein phosphatase 1 binding / eukaryotic 48S preinitiation complex / DNA catabolic process / Formation of the ternary complex, and subsequently, the 43S complex / Translation initiation complex formation / Ribosomal scanning and start codon recognition / cytoskeletal motor activator activity / myosin phosphatase activity / negative regulation of phosphoprotein phosphatase activity / protein serine/threonine phosphatase activity / glycogen metabolic process / detection of maltose stimulus / maltose transport complex / negative regulation of PERK-mediated unfolded protein response / protein-serine/threonine phosphatase / tropomyosin binding / mesenchyme migration / troponin I binding / myosin heavy chain binding / entrainment of circadian clock by photoperiod / protein phosphatase activator activity / phosphatase activity / filamentous actin / actin filament bundle / carbohydrate transport / positive regulation of phosphoprotein phosphatase activity / phosphoprotein phosphatase activity / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle assembly / Response of EIF2AK4 (GCN2) to amino acid deficiency / carbohydrate transmembrane transporter activity / skeletal muscle myofibril / L13a-mediated translational silencing of Ceruloplasmin expression / GTP hydrolysis and joining of the 60S ribosomal subunit / maltose binding / actin monomer binding / maltose transport / maltodextrin transmembrane transport / intrinsic apoptotic signaling pathway in response to endoplasmic reticulum stress / skeletal muscle fiber development / stress granule assembly / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / stress fiber / titin binding / translational initiation / translation initiation factor activity / cellular response to amino acid starvation / actin filament polymerization / response to endoplasmic reticulum stress / ATP-binding cassette (ABC) transporter complex / protein dephosphorylation / Downregulation of TGF-beta receptor signaling / cell chemotaxis / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / circadian regulation of gene expression / ABC-family proteins mediated transport / PKR-mediated signaling / regulation of circadian rhythm / cytoplasmic stress granule / calcium-dependent protein binding / cellular response to UV / positive regulation of canonical Wnt signaling pathway / lamellipodium / ribosome binding / nuclear envelope / actin binding
Similarity search - Function
Protein phosphatase 1, regulatory subunit 15A/B, C-terminal / Phosphatase-1 catalytic subunit binding region / Deoxyribonuclease I / Deoxyribonuclease I, active site / Deoxyribonuclease I, conservied site / Deoxyribonuclease I signature 2. / Deoxyribonuclease I signature 1. / deoxyribonuclease I / Serine-threonine protein phosphatase, N-terminal / Serine-threonine protein phosphatase N-terminal domain ...Protein phosphatase 1, regulatory subunit 15A/B, C-terminal / Phosphatase-1 catalytic subunit binding region / Deoxyribonuclease I / Deoxyribonuclease I, active site / Deoxyribonuclease I, conservied site / Deoxyribonuclease I signature 2. / Deoxyribonuclease I signature 1. / deoxyribonuclease I / Serine-threonine protein phosphatase, N-terminal / Serine-threonine protein phosphatase N-terminal domain / IF2a, S1-like domain / Translation initiation factor 2, alpha subunit / Translation initiation factor 2, alpha subunit, middle domain superfamily / Translation initiation factor 2, alpha subunit, C-terminal / Eukaryotic translation initiation factor 2 alpha subunit / Serine/threonine specific protein phosphatases signature. / Protein phosphatase 2A homologues, catalytic domain. / Serine/threonine-specific protein phosphatase/bis(5-nucleosyl)-tetraphosphatase / Endonuclease/exonuclease/phosphatase / Endonuclease/Exonuclease/phosphatase family / Endonuclease/exonuclease/phosphatase superfamily / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase / Metallo-dependent phosphatase-like / S1 domain profile. / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Bacterial extracellular solute-binding protein / Ribosomal protein S1-like RNA-binding domain / Actin / S1 RNA binding domain / Actin family / Actin / S1 domain / ATPase, nucleotide binding domain / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / : / Protein phosphatase 1 regulatory subunit 15A / Deoxyribonuclease-1 / Eukaryotic translation initiation factor 2 subunit 1 / Maltose/maltodextrin-binding periplasmic protein / Serine/threonine-protein phosphatase PP1-alpha catalytic subunit / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesHomo sapiens (human)
Oryctolagus cuniculus (rabbit)
Escherichia coli (E. coli)
Bos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.96 Å
AuthorsYan, Y. / Hardwick, S. / Ron, D.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome TrustWT 2008/Z/16/Z United Kingdom
CitationJournal: Nat Struct Mol Biol / Year: 2021
Title: Higher-order phosphatase-substrate contacts terminate the integrated stress response.
Authors: Yahui Yan / Heather P Harding / David Ron /
Abstract: Many regulatory PPP1R subunits join few catalytic PP1c subunits to mediate phosphoserine and phosphothreonine dephosphorylation in metazoans. Regulatory subunits engage the surface of PP1c, locally ...Many regulatory PPP1R subunits join few catalytic PP1c subunits to mediate phosphoserine and phosphothreonine dephosphorylation in metazoans. Regulatory subunits engage the surface of PP1c, locally affecting flexible access of the phosphopeptide to the active site. However, catalytic efficiency of holophosphatases towards their phosphoprotein substrates remains unexplained. Here we present a cryo-EM structure of the tripartite PP1c-PPP1R15A-G-actin holophosphatase that terminates signaling in the mammalian integrated stress response (ISR) in the pre-dephosphorylation complex with its substrate, translation initiation factor 2α (eIF2α). G-actin, whose essential role in eIF2α dephosphorylation is supported crystallographically, biochemically and genetically, aligns the catalytic and regulatory subunits, creating a composite surface that engages the N-terminal domain of eIF2α to position the distant phosphoserine-51 at the active site. Substrate residues that mediate affinity for the holophosphatase also make critical contacts with eIF2α kinases. Thus, a convergent process of higher-order substrate recognition specifies functionally antagonistic phosphorylation and dephosphorylation in the ISR.
History
DepositionMar 24, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 29, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 20, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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 / _citation_author.name / _em_admin.last_update

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Structure visualization

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  • Deposited structure unit
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  • EMDB-12665
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Structure viewerMolecule:
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Assembly

Deposited unit
E: Eukaryotic translation initiation factor 2 subunit 1
B: Serine/threonine-protein phosphatase PP1-alpha catalytic subunit
A: Actin, alpha skeletal muscle, intermediate form
D: Deoxyribonuclease-1
C: Protein phosphatase 1 regulatory subunit 15A,Maltose/maltodextrin-binding periplasmic protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)176,5778
Polymers175,5905
Non-polymers9873
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, A complex has one copy of each following component: PP1, PPP1R15A, G-actin, DNase I, eIF2alpha.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10630 Å2
ΔGint-53 kcal/mol
Surface area52090 Å2
MethodPISA

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Components

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Protein , 5 types, 5 molecules EBADC

#1: Protein Eukaryotic translation initiation factor 2 subunit 1 / Eukaryotic translation initiation factor 2 subunit alpha / eIF-2-alpha / eIF-2A / eIF-2alpha


Mass: 21817.863 Da / Num. of mol.: 1 / Mutation: phosphorylated Ser51
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EIF2S1, EIF2A / Production host: Escherichia coli (E. coli) / References: UniProt: P05198
#2: Protein Serine/threonine-protein phosphatase PP1-alpha catalytic subunit / PP-1A


Mass: 33647.621 Da / Num. of mol.: 1 / Mutation: D64A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oryctolagus cuniculus (rabbit) / Gene: PPP1CA, PPP1A / Production host: Escherichia coli (E. coli)
References: UniProt: P62139, protein-serine/threonine phosphatase
#3: Protein Actin, alpha skeletal muscle, intermediate form


Mass: 41862.613 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P68135
#4: Protein Deoxyribonuclease-1 / Deoxyribonuclease I / DNase I


Mass: 29092.574 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: P00639, deoxyribonuclease I
#5: Protein Protein phosphatase 1 regulatory subunit 15A,Maltose/maltodextrin-binding periplasmic protein / Growth arrest and DNA damage-inducible protein GADD34 / Myeloid differentiation primary response ...Growth arrest and DNA damage-inducible protein GADD34 / Myeloid differentiation primary response protein MyD116 homolog / MMBP / Maltodextrin-binding protein / Maltose-binding protein / MBP


Mass: 49169.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: PPP1R15A, GADD34, malE, b4034, JW3994 / Strain: K12 / Production host: Escherichia coli (E. coli) / References: UniProt: O75807, UniProt: P0AEX9

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Sugars , 1 types, 1 molecules

#6: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE

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Non-polymers , 2 types, 2 molecules

#7: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM

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Details

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: pre-dephosphorylation complex of phosphorylated eIF2alpha_2-187 with trapped holophosphatase (PP1A_D64A/PPP1R15A_553-624/G-actin)
Type: COMPLEX
Details: One copy of each component was present in the complex: phosphorylated eIF2alpha_2-187, PP1A_D64A, PPP1R15A_553-624, G-actin and DNase I. The full complex was purified by size exclusion chromatography.
Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.181 MDa / Experimental value: YES
Buffer solutionpH: 7.4
Details: 0.22mM Triton X-100 was added into the solution before plunging.
Buffer component
IDConc.NameBuffer-ID
110 mMTris1
2150 mMNaCl1
31 mMMnCl21
41 mMCaCl21
50.2 mMTCEP1
60.2 mMATP1
70.22 mMTriton X-1001
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: current 25mA at Pelco EasiGLOW / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: -2800 nm / Nominal defocus min: -1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 46.84 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4025
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV / Phase plate: VOLTA PHASE PLATE

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Processing

EM software
IDNameCategory
1Warpparticle selection
2EPUimage acquisition
4WarpCTF correction
5cryoSPARCCTF correction
8UCSF Chimeramodel fitting
9Cootmodel fitting
11cryoSPARCinitial Euler assignment
12cryoSPARCfinal Euler assignment
13cryoSPARCclassification
14cryoSPARC3D reconstruction
15PHENIXmodel refinement
CTF correctionDetails: Warp estimated the CTF parameters and passed them on to CryoSPARC to perform CTF correction.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 132495
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 60413 / Details: non-uniform refinement / Symmetry type: POINT
Atomic model buildingB value: 47 / Protocol: OTHER / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
12A42

2a42

A1
22A42

2a42

D1
34MOV

4mov

B1
41KL9

1kl9

E1

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