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- PDB-6h8c: Structure of the human GABARAPL2 protein in complex with the UBA5... -

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Basic information

Entry
Database: PDB / ID: 6h8c
TitleStructure of the human GABARAPL2 protein in complex with the UBA5 LIR motif
Components
  • Gamma-aminobutyric acid receptor-associated protein-like 2
  • Ubiquitin-like modifier-activating enzyme 5
KeywordsSIGNALING PROTEIN / GABARAPL2 / UBA5 LIR motif / protein complex
Function / homology
Function and homology information


UFM1 activating enzyme activity / protein ufmylation / protein K69-linked ufmylation / megakaryocyte differentiation / negative regulation of proteasomal protein catabolic process / protein localization to endoplasmic reticulum / GABA receptor binding / intra-Golgi vesicle-mediated transport / regulation of intracellular estrogen receptor signaling pathway / cellular response to nitrogen starvation ...UFM1 activating enzyme activity / protein ufmylation / protein K69-linked ufmylation / megakaryocyte differentiation / negative regulation of proteasomal protein catabolic process / protein localization to endoplasmic reticulum / GABA receptor binding / intra-Golgi vesicle-mediated transport / regulation of intracellular estrogen receptor signaling pathway / cellular response to nitrogen starvation / phosphatidylethanolamine binding / positive regulation of ATP-dependent activity / TBC/RABGAPs / reticulophagy / neuromuscular process / Macroautophagy / beta-tubulin binding / autophagosome membrane / autophagosome maturation / autophagosome assembly / autophagosome / localization / response to endoplasmic reticulum stress / erythrocyte differentiation / SNARE binding / autophagy / Antigen processing: Ubiquitination & Proteasome degradation / protein transport / ATPase binding / cytoplasmic vesicle / microtubule binding / Golgi membrane / intracellular membrane-bounded organelle / ubiquitin protein ligase binding / endoplasmic reticulum membrane / Golgi apparatus / protein homodimerization activity / zinc ion binding / ATP binding / nucleus / cytoplasm / cytosol
Similarity search - Function
Autophagy protein Atg8 ubiquitin-like / Autophagy protein Atg8 ubiquitin like / D-isomer specific 2-hydroxyacid dehydrogenase, NAD-binding domain conserved site 1 / ThiF/MoeB/HesA family / Ubiquitin-activating enzyme / THIF-type NAD/FAD binding fold / ThiF family / Phosphatidylinositol 3-kinase Catalytic Subunit; Chain A, domain 1 / Ubiquitin-like (UB roll) / Ubiquitin-like domain superfamily ...Autophagy protein Atg8 ubiquitin-like / Autophagy protein Atg8 ubiquitin like / D-isomer specific 2-hydroxyacid dehydrogenase, NAD-binding domain conserved site 1 / ThiF/MoeB/HesA family / Ubiquitin-activating enzyme / THIF-type NAD/FAD binding fold / ThiF family / Phosphatidylinositol 3-kinase Catalytic Subunit; Chain A, domain 1 / Ubiquitin-like (UB roll) / Ubiquitin-like domain superfamily / Roll / Alpha Beta
Similarity search - Domain/homology
Gamma-aminobutyric acid receptor-associated protein-like 2 / Ubiquitin-like modifier-activating enzyme 5
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodSOLUTION NMR / torsion angle dynamics
AuthorsHuber, J. / Loehr, F. / Gruber, J. / Akutsu, M. / Guentert, P. / Doetsch, V. / Rogov, V.V.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Federal Ministry for Education and ResearchSFB 1177 Germany
CitationJournal: Autophagy / Year: 2020
Title: An atypical LIR motif within UBA5 (ubiquitin like modifier activating enzyme 5) interacts with GABARAP proteins and mediates membrane localization of UBA5.
Authors: Huber, J. / Obata, M. / Gruber, J. / Akutsu, M. / Lohr, F. / Rogova, N. / Guntert, P. / Dikic, I. / Kirkin, V. / Komatsu, M. / Dotsch, V. / Rogov, V.V.
History
DepositionAug 2, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 1, 2019Provider: repository / Type: Initial release
Revision 1.1May 8, 2019Group: Data collection / Database references
Category: citation / database_PDB_rev ...citation / database_PDB_rev / database_PDB_rev_record / pdbx_nmr_software
Item: _citation.page_first / _citation.page_last / _pdbx_nmr_software.name
Revision 1.2Jul 17, 2019Group: Data collection / Derived calculations
Category: pdbx_struct_sheet_hbond / struct_conf / struct_sheet_range
Item: _pdbx_struct_sheet_hbond.range_1_auth_seq_id / _pdbx_struct_sheet_hbond.range_1_label_seq_id ..._pdbx_struct_sheet_hbond.range_1_auth_seq_id / _pdbx_struct_sheet_hbond.range_1_label_seq_id / _pdbx_struct_sheet_hbond.range_2_auth_comp_id / _pdbx_struct_sheet_hbond.range_2_auth_seq_id / _pdbx_struct_sheet_hbond.range_2_label_comp_id / _pdbx_struct_sheet_hbond.range_2_label_seq_id / _struct_conf.end_auth_comp_id / _struct_conf.end_auth_seq_id / _struct_conf.end_label_comp_id / _struct_conf.end_label_seq_id / _struct_conf.pdbx_PDB_helix_length / _struct_sheet_range.end_auth_comp_id / _struct_sheet_range.end_auth_seq_id / _struct_sheet_range.end_label_comp_id / _struct_sheet_range.end_label_seq_id
Revision 1.3Jan 29, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID
Revision 1.4May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Gamma-aminobutyric acid receptor-associated protein-like 2
B: Ubiquitin-like modifier-activating enzyme 5


Theoretical massNumber of molelcules
Total (without water)15,6402
Polymers15,6402
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: isothermal titration calorimetry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1870 Å2
ΔGint-9 kcal/mol
Surface area8710 Å2
MethodPISA
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 200target function
RepresentativeModel #1closest to the average

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Components

#1: Protein Gamma-aminobutyric acid receptor-associated protein-like 2 / GABA(A) receptor-associated protein-like 2 / Ganglioside expression factor 2 / GEF-2 / General ...GABA(A) receptor-associated protein-like 2 / Ganglioside expression factor 2 / GEF-2 / General protein transport factor p16 / Golgi-associated ATPase enhancer of 16 kDa / GATE-16 / MAP1 light chain 3-related protein


Mass: 13467.532 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Gamma-aminobutyric acid receptor-associated protein-like 2 (GABARAPL2) residues 3-116, residues 1-2 are expressing tag
Source: (gene. exp.) Homo sapiens (human) / Gene: GABARAPL2, FLC3A, GEF2 / Plasmid: pET39 / Details (production host): Ub19 leader / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): NEB T7 Express / References: UniProt: P60520
#2: Protein/peptide Ubiquitin-like modifier-activating enzyme 5 / Ubiquitin-activating enzyme 5 / ThiFP1 / UFM1-activating enzyme / Ubiquitin-activating enzyme E1 ...Ubiquitin-activating enzyme 5 / ThiFP1 / UFM1-activating enzyme / Ubiquitin-activating enzyme E1 domain-containing protein 1


Mass: 2172.348 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: UFM1-activating enzyme 5 (UBA5) LIR/UFIM containing peptide (resides 333-348). First 3 residues (Gly-Ala-Met) are expression tag.
Source: (gene. exp.) Homo sapiens (human) / Gene: UBA5, UBE1DC1 / Plasmid: pET39 / Details (production host): Ub19 leader / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): NEB T7 Express / References: UniProt: Q9GZZ9

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic52D 1H-15N HSQC
122isotropic52D 1H-15N HSQC
131isotropic12D 1H-13C HSQC
142isotropic12D 1H-13C HSQC
151isotropic33D HN(CA)CB
162isotropic33D HN(CA)CB
181isotropic43D (HCA)CO(CA)NH
172isotropic43D (HCA)CO(CA)NH
1141isotropic23D H(CC)(CO)NH-TOCSY
1132isotropic23D H(CC)(CO)NH-TOCSY
1121isotropic23D (H)CC(CO)NH-TOCSY
1112isotropic23D (H)CC(CO)NH-TOCSY
1101isotropic13D 1H-15N NOESY
192isotropic13D 1H-15N NOESY
1161isotropic13D 1H-13C NOESY
1182isotropic13D 1H-13C NOESY
1171isotropic23D NOESY-[13C,1H]-HSQC 13C/15N filtered in F1
1152isotropic23D NOESY-[13C,1H]-HSQC 13C/15N filtered in F1

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
solution10.6 mM [U-99% 13C; U-99% 15N] GABARAPL2, 1.0 mM No Ubiquitin-like modifier-activating enzyme 5 (UBA5) LIR motif, 50 mM No sodium phosphate, 100 mM No sodium chloride, 4.6 mM No sodium azide, 95% H2O/5% D2OGABARAPL2, [U-99% 13C; U-99% 15N], 0.6 mM; UBA5 LIR 1.0 mM; sodium phosphate 50 mM; sodium chloride 100 mM; sodium azide 4.6 mM; protease inhibitor cocktail 1 mM; DSS 0.3 mM; H2O 95%; D2O 5%13C,15N GABARAPL2 - UBA5 LIR/UFIM95% H2O/5% D2O
solution21.0 mM No GABARAPL2, 0.6 mM [U-99% 13C; U-99% 15N] Ubiquitin-like modifier-activating enzyme 5 (UBA5) LIR motif, 50 mM No sodium phosphate, 100 mM No sodium chloride, 4.6 mM No sodium azide, 95% H2O/5% D2OUBA5 LIR, [U-99% 13C; U-99% 15N], 0.6 mM; GABARAPL2 1.0 mM; sodium phosphate 50 mM; sodium chloride 100 mM; sodium azide 4.6 mM; protease inhibitor cocktail 1 mM; DSS 0.3 mM; H2O 95%; D2O 5%13C,15N UBA5 LIR/UFIM - GABARAPL295% H2O/5% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.6 mMGABARAPL2[U-99% 13C; U-99% 15N]1
1.0 mMUbiquitin-like modifier-activating enzyme 5 (UBA5) LIR motifNo1
50 mMsodium phosphateNo1
100 mMsodium chlorideNo1
4.6 mMsodium azideNo1
1.0 mMGABARAPL2No2
0.6 mMUbiquitin-like modifier-activating enzyme 5 (UBA5) LIR motif[U-99% 13C; U-99% 15N]2
50 mMsodium phosphateNo2
100 mMsodium chlorideNo2
4.6 mMsodium azideNo2
Sample conditionsIonic strength: 100 mM / Ionic strength err: 5 / Label: conditions_1 / pH: 7.0 / PH err: 0.1 / Pressure: AMBIENT atm / Temperature: 298 K / Temperature err: 0.2

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker Bruker Avance 950BrukerBruker Avance 950 9501
Bruker Bruker Avance 900BrukerBruker Avance 900 9002
Bruker Bruker Avance 800BrukerBruker Avance 800 8003
Bruker Bruker Avance 700BrukerBruker Avance 700 7004
Bruker Bruker Avance 600BrukerBruker Avance 600 6005
Bruker Bruker Avance 500BrukerBruker Avance 500 5006

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Processing

NMR software
NameVersionDeveloperClassification
TopSpin3.1Bruker Biospincollection
Sparky1.114Goddardchemical shift assignment
CYANA3.97Guntert, Mumenthaler and Wuthrichpeak picking
CYANA3.97Guntert, Mumenthaler and Wuthrichstructure calculation
OPALLuginbuhl, Guntert, Billeter and Wuthrichrefinement
RefinementMethod: torsion angle dynamics / Software ordinal: 5 / Details: AMBER94
NMR representativeSelection criteria: closest to the average
NMR ensembleConformer selection criteria: target function / Conformers calculated total number: 200 / Conformers submitted total number: 20

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