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- PDB-8tgf: Crystal structure of cEPG5 LIR/LGG-1 complex -

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Basic information

Entry
Database: PDB / ID: 8tgf
TitleCrystal structure of cEPG5 LIR/LGG-1 complex
Components
  • Ectopic P granules protein 5
  • Protein lgg-1
KeywordsSIGNALING PROTEIN
Function / homology
Function and homology information


Macroautophagy / dauer larval development / xenophagy / positive regulation of autophagosome assembly / negative regulation of autophagosome assembly / GABA receptor binding / plasma membrane repair / cellular response to toxic substance / programmed cell death / phosphatidylethanolamine binding ...Macroautophagy / dauer larval development / xenophagy / positive regulation of autophagosome assembly / negative regulation of autophagosome assembly / GABA receptor binding / plasma membrane repair / cellular response to toxic substance / programmed cell death / phosphatidylethanolamine binding / phagophore assembly site / cellular response to nitrogen starvation / necroptotic process / autophagosome membrane / autophagosome assembly / autophagosome maturation / mitophagy / lysosomal lumen / autophagosome / determination of adult lifespan / macroautophagy / autophagy / phagocytic vesicle membrane / response to heat / perikaryon / mitochondrial outer membrane / defense response to Gram-positive bacterium / neuron projection / neuronal cell body / dendrite / ubiquitin protein ligase binding / nucleus / plasma membrane / cytoplasm
Similarity search - Function
: / Autophagy protein Atg8 ubiquitin-like / Autophagy protein Atg8 ubiquitin like / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
ACETATE ION / Protein lgg-1 / Ectopic P granules protein 5
Similarity search - Component
Biological speciesCaenorhabditis elegans (invertebrata)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.6 Å
AuthorsCheung, Y.W.S. / Yip, C.K.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
CitationJournal: Autophagy / Year: 2025
Title: Structure of the human autophagy factor EPG5 and the molecular basis of its conserved mode of interaction with Atg8-family proteins.
Authors: Yiu Wing Sunny Cheung / Sung-Eun Nam / Gage M J Fairlie / Karlton Scheu / Jennifer M Bui / Hannah R Shariati / Jörg Gsponer / Calvin K Yip /
Abstract: The multi-step macroautophagy/autophagy process ends with the cargo-laden autophagosome fusing with the lysosome to deliver the materials to be degraded. The metazoan-specific autophagy factor EPG5 ...The multi-step macroautophagy/autophagy process ends with the cargo-laden autophagosome fusing with the lysosome to deliver the materials to be degraded. The metazoan-specific autophagy factor EPG5 plays a crucial role in this step by enforcing fusion specificity and preventing mistargeting. How EPG5 exerts its critical function and how its deficiency leads to diverse phenotypes of the rare multi-system disorder Vici syndrome are not fully understood. Here, we report the first structure of human EPG5 (HsEPG5) determined by cryo-EM and AlphaFold2 modeling. Our structure revealed that HsEPG5 is constructed from helical bundles analogous to tethering factors in membrane trafficking pathways but contains a unique protruding thumb domain positioned adjacent to the atypical tandem LIR motifs involved in interaction with the GABARAP subfamily of Atg8-family proteins. Our NMR spectroscopic, molecular dynamics simulations and AlphaFold modeling studies showed that the HsEPG5 tandem LIR motifs only bind the canonical LIR docking site (LDS) on GABARAP without engaging in multivalent interaction. Our co-immunoprecipitation analysis further indicated that full-length HsEPG5-GABARAP interaction is mediated primarily by LIR1. Finally, our biochemical affinity isolation, X-ray crystallographic analysis, affinity measurement, and AlphaFold modeling demonstrated that this mode of binding is observed between EPG-5 and its Atg8-family proteins LGG-1 and LGG-2. Collectively our work generated novel insights into the structural properties of EPG5 and how it potentially engages with the autophagosome to confer fusion specificity.: ATG: autophagy related; CSP: chemical shift perturbation; eGFP: enhanced green fluoresent protein; EM: electron microscopy; EPG5: ectopic P-granules 5 autophagy tethering factor; GST: glutathione S-transferase; HP: hydrophobic pocket; HSQC: heteronuclear single-quantum correlation; ITC: isothermal titration calorimetry; LDS: LC3 docking site; LIR: LC3-interacting region; MD: molecular dynamics; NMR: nuclear magnetic resonance; TEV: tobacco etch virus.
History
DepositionJul 12, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 17, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 22, 2025Group: Database references / Structure summary / Category: citation / citation_author / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _pdbx_entry_details.has_protein_modification
Revision 1.2Jan 29, 2025Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.3May 28, 2025Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Protein lgg-1
B: Ectopic P granules protein 5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,2713
Polymers16,2122
Non-polymers591
Water1,17165
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)33.991, 37.952, 45.177
Angle α, β, γ (deg.)90.000, 100.102, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Protein Protein lgg-1


Mass: 14933.061 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: lgg-1, atg-8.1, C32D5.9 / Production host: Escherichia coli (E. coli) / References: UniProt: Q09490
#2: Protein/peptide Ectopic P granules protein 5


Mass: 1279.265 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Caenorhabditis elegans (invertebrata) / References: UniProt: Q18892
#3: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H3O2
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 65 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 1.79 Å3/Da / Density % sol: 31.49 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 8 / Details: 0.1 M sodium acetate pH 4.6, 21% (w/v) PEG 6000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.2 / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Oct 16, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.6→44.48 Å / Num. obs: 14650 / % possible obs: 96.93 % / Redundancy: 3.3 % / Biso Wilson estimate: 14.59 Å2 / Rmerge(I) obs: 0.0364 / Net I/σ(I): 25.12
Reflection shellResolution: 1.6→1.657 Å / Rmerge(I) obs: 0.1156 / Num. unique obs: 1406

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Processing

Software
NameVersionClassification
DIALSdata reduction
Aimless0.7.9data scaling
PHASER2.8.2phasing
PHENIX1.14_3260refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5AZF

5azf


Resolution: 1.6→44.48 Å / SU ML: 0.1491 / Cross valid method: THROUGHOUT / σ(F): 1.47 / Phase error: 22.0927 / Stereochemistry target values: CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2134 724 4.94 %
Rwork0.1743 13923 -
obs0.1762 14647 96.94 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 22.52 Å2
Refinement stepCycle: LAST / Resolution: 1.6→44.48 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1002 0 4 65 1071
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01011036
X-RAY DIFFRACTIONf_angle_d1.09021393
X-RAY DIFFRACTIONf_chiral_restr0.0698146
X-RAY DIFFRACTIONf_plane_restr0.0075178
X-RAY DIFFRACTIONf_dihedral_angle_d2.6402872
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.6-1.720.22951360.18132756X-RAY DIFFRACTION97.18
1.72-1.90.23021490.17422790X-RAY DIFFRACTION97.54
1.9-2.170.2111560.16612720X-RAY DIFFRACTION96.06
2.17-2.740.21911320.17952783X-RAY DIFFRACTION96.56
2.74-44.480.20471510.17342874X-RAY DIFFRACTION97.36
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.17727153718-0.0240872748873-0.03635064295272.44772583851-0.02622278822182.598325596530.0181722663387-0.00216498585229-0.201894310231-0.005255671210740.01533296847080.1292034082750.219192124217-0.106313915491-0.03899580668640.0990898033278-0.00708522175863-0.001933040566680.0527254604252-0.005898367361810.08726761165113.60940698152.713089554728.95489426179
25.3351982118-0.96751008405-1.86548061295.41257433990.5541517413231.6107160325-0.0137674789692-0.394499287901-0.0267538713360.298479267176-0.0300418473696-0.134123847085-0.0150443695715-0.07694202300720.02741733405060.284066698772-0.03153141680340.0446567968160.139468226506-0.04998280921410.15380051735114.93342266515.453911920620.6892308196
31.38390554231-1.06440470151-1.480716390624.79025103076-0.9230820215134.20751683389-0.01123918356090.04046402607410.0474629926332-0.004164270155450.1202502118650.190857183574-0.26191179644-0.192220721406-0.03398863511190.12288898302-0.009004314366770.007083707849180.07642791404780.01633395584610.081521564504415.113648545817.69987170125.1822685006
43.163418643611.42477812018-0.08683343008573.949433561160.08914972566252.19931690488-0.0675718438115-0.09460611631330.4110946400170.1673603589920.0450451664810.215738564879-0.246436245442-0.2036989227860.05873519639410.1301300690770.02127611198170.03061785885250.1123425778040.004972661442370.072046875970710.25375213469.7994142189414.7183129304
50.8519884621960.2474009957120.567617320682.99296287757-0.8579027592224.899091418340.0501460316585-0.0032419430420.09817661026920.4692706856770.02389928979930.394921304139-0.216279842245-0.336366252128-0.2991184225970.3474707146020.03601913382670.05016578632210.1191829355910.09631850292410.06696010327399.19008672772.9126190702321.3715320134
61.323260967770.308434562991-0.09594380179050.35278905414-0.4922800199871.01903432043-0.0459957656783-0.661396854004-0.00630501852840.5390628479090.04934621584820.733850377147-0.123262412657-0.7564026056180.01311977742080.2425212489350.04983508020430.07757813430410.488288533340.1080792016320.3881825623910.5171901118112.2322410325918.9217082588
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 78 through 113 )
2X-RAY DIFFRACTION2chain 'B' and (resid 505 through 513 )
3X-RAY DIFFRACTION3chain 'A' and (resid 1 through 24 )
4X-RAY DIFFRACTION4chain 'A' and (resid 25 through 56 )
5X-RAY DIFFRACTION5chain 'A' and (resid 57 through 66 )
6X-RAY DIFFRACTION6chain 'A' and (resid 67 through 77 )

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