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- PDB-7t92: Structure of the peroxisomal retro-translocon formed by a heterot... -

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Basic information

Entry
Database: PDB / ID: 7t92
TitleStructure of the peroxisomal retro-translocon formed by a heterotrimeric ubiquitin ligase complex
Components
  • Fab heavy chain
  • Fab light chain
  • Peroxin-10
  • Peroxin-12
  • Peroxin-2
KeywordsTRANSLOCASE / peroxisome / retro-translocon / ubiquitin ligase
Function / homology
Function and homology information


: / protein import into peroxisome matrix / : / peroxisome / zinc ion binding / metal ion binding
Similarity search - Function
Pex, N-terminal / Peroxisome assembly protein 12 / Pex2 / Pex12 amino terminal region / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / Ring finger / Zinc finger RING-type profile. / Zinc finger, RING-type / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
CHOLESTEROL / 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / RING-type domain-containing protein / Pex2_Pex12 domain-containing protein / Pex2_Pex12 domain-containing protein
Similarity search - Component
Biological speciesThermothelomyces thermophilus ATCC 42464 (fungus)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsPeiqiang, F. / Tom, R.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2022
Title: A peroxisomal ubiquitin ligase complex forms a retrotranslocation channel.
Authors: Peiqiang Feng / Xudong Wu / Satchal K Erramilli / Joao A Paulo / Pawel Knejski / Steven P Gygi / Anthony A Kossiakoff / Tom A Rapoport /
Abstract: Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health. Luminal peroxisomal proteins are imported from the cytosol by mobile receptors, which ...Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health. Luminal peroxisomal proteins are imported from the cytosol by mobile receptors, which then recycle back to the cytosol by a poorly understood process. Recycling requires receptor modification by a membrane-embedded ubiquitin ligase complex comprising three RING finger domain-containing proteins (Pex2, Pex10 and Pex12). Here we report a cryo-electron microscopy structure of the ligase complex, which together with biochemical and in vivo experiments reveals its function as a retrotranslocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that co-assemble into an open channel. The three ring finger domains form a cytosolic tower, with ring finger 2 (RF2) positioned above the channel pore. We propose that the N terminus of a recycling receptor is inserted from the peroxisomal lumen into the pore and monoubiquitylated by RF2 to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitylated by the concerted action of RF10 and RF12 and degraded. This polyubiquitylation pathway also maintains the homeostasis of other peroxisomal import factors. Our results clarify a crucial step during peroxisomal protein import and reveal why mutations in the ligase complex cause human disease.
History
DepositionDec 17, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 6, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2022Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jul 27, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Feb 28, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Peroxin-12
A: Peroxin-2
C: Peroxin-10
H: Fab heavy chain
L: Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)174,13130
Polymers160,4705
Non-polymers13,66125
Water00
1


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

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Components

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Protein , 3 types, 3 molecules BAC

#1: Protein Peroxin-12


Mass: 49369.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermothelomyces thermophilus ATCC 42464 (fungus)
Strain: ATCC 42464 / BCRC 31852 / DSM 1799 / Gene: MYCTH_2053677 / Production host: Komagataella pastoris (fungus) / References: UniProt: G2Q5N0
#2: Protein Peroxin-2


Mass: 38493.715 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermothelomyces thermophilus ATCC 42464 (fungus)
Strain: ATCC 42464 / BCRC 31852 / DSM 1799 / Gene: MYCTH_2294472 / Production host: Komagataella pastoris (fungus) / References: UniProt: G2Q1C9
#3: Protein Peroxin-10


Mass: 48952.234 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermothelomyces thermophilus ATCC 42464 (fungus)
Strain: ATCC 42464 / BCRC 31852 / DSM 1799 / Gene: MYCTH_2294231 / Production host: Komagataella pastoris (fungus) / References: UniProt: G2Q0E2

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Antibody , 2 types, 2 molecules HL

#4: Antibody Fab heavy chain


Mass: 12518.806 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#5: Antibody Fab light chain


Mass: 11135.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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Non-polymers , 3 types, 25 molecules

#6: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Zn
#7: Chemical
ChemComp-LBN / 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / (2R)-2-[(9Z)-9-Octadecenoyloxy]-3-(palmitoyloxy)propyl 2-(trimethylammonio)ethyl phosphate


Mass: 760.076 Da / Num. of mol.: 15 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM
#8: Chemical
ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C27H46O

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Details

Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Peroxisomal heterotrimeric ubiquitin ligase complex bound with FabCOMPLEX#1-#50RECOMBINANT
2Peroxin12, Peroxin2, Peroxin10COMPLEX#1-#31RECOMBINANT
3Fab heavy chain, Fab light chainCOMPLEX#4-#51RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Thermothelomyces thermophilus ATCC 42464 (fungus)573729
23synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Komagataella pastoris (fungus)4922
23Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / 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: 1000 nm
Image recordingElectron dose: 52 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 121644 / Symmetry type: POINT
RefinementHighest resolution: 3.1 Å / Cross valid method: NONE
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00599842
ELECTRON MICROSCOPYf_angle_d0.704513320
ELECTRON MICROSCOPYf_chiral_restr0.07961453
ELECTRON MICROSCOPYf_plane_restr0.00491636
ELECTRON MICROSCOPYf_dihedral_angle_d11.62811485

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