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- PDB-8oif: Structure of the UBE1L activating enzyme bound to ISG15 and UBE2L6 -

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Basic information

Entry
Database: PDB / ID: 8oif
TitleStructure of the UBE1L activating enzyme bound to ISG15 and UBE2L6
Components
  • Ubiquitin-like modifier-activating enzyme 7
  • Ubiquitin-like protein ISG15
  • Ubiquitin/ISG15-conjugating enzyme E2 L6
KeywordsANTIVIRAL PROTEIN / ubiquitin-like / ISG15 / interferon-stimulated genes / antiviral
Function / homology
Function and homology information


ISG15 activating enzyme activity / ISG15 transferase activity / Ligases; Forming carbon-sulfur bonds; Acid-thiol ligases / positive regulation of protein oligomerization / ISG15-protein conjugation / regulation of type II interferon production / protein localization to mitochondrion / NS1 Mediated Effects on Host Pathways / response to type I interferon / negative regulation of type I interferon-mediated signaling pathway ...ISG15 activating enzyme activity / ISG15 transferase activity / Ligases; Forming carbon-sulfur bonds; Acid-thiol ligases / positive regulation of protein oligomerization / ISG15-protein conjugation / regulation of type II interferon production / protein localization to mitochondrion / NS1 Mediated Effects on Host Pathways / response to type I interferon / negative regulation of type I interferon-mediated signaling pathway / negative regulation of viral genome replication / E2 ubiquitin-conjugating enzyme / RSV-host interactions / ubiquitin conjugating enzyme activity / positive regulation of interleukin-10 production / positive regulation of bone mineralization / ubiquitin ligase complex / negative regulation of protein ubiquitination / positive regulation of interferon-beta production / positive regulation of erythrocyte differentiation / ubiquitin binding / integrin-mediated signaling pathway / Negative regulators of DDX58/IFIH1 signaling / Termination of translesion DNA synthesis / response to virus / DDX58/IFIH1-mediated induction of interferon-alpha/beta / PKR-mediated signaling / protein modification process / ISG15 antiviral mechanism / modification-dependent protein catabolic process / protein polyubiquitination / positive regulation of type II interferon production / ubiquitin-protein transferase activity / protein tag activity / Interferon alpha/beta signaling / integrin binding / Antigen processing: Ubiquitination & Proteasome degradation / ubiquitin-dependent protein catabolic process / defense response to virus / protein ubiquitination / defense response to bacterium / Amyloid fiber formation / innate immune response / ubiquitin protein ligase binding / DNA damage response / SARS-CoV-2 activates/modulates innate and adaptive immune responses / extracellular region / nucleoplasm / ATP binding / nucleus / cytosol / cytoplasm
Similarity search - Function
: / Ubiquitin-activating enzyme E1 / Ubiquitin-activating enzyme E1, C-terminal / Ubiquitin-activating enzyme E1, FCCH domain / Ubiquitin-activating enzyme E1, four-helix bundle / Ubiquitin-activating enzyme E1, C-terminal domain superfamily / Ubiquitin-activating enzyme E1, SCCH domain / Ubiquitin-activating enzyme E1, FCCH domain superfamily / Ubiquitin fold domain / Ubiquitin-activating enzyme E1 FCCH domain ...: / Ubiquitin-activating enzyme E1 / Ubiquitin-activating enzyme E1, C-terminal / Ubiquitin-activating enzyme E1, FCCH domain / Ubiquitin-activating enzyme E1, four-helix bundle / Ubiquitin-activating enzyme E1, C-terminal domain superfamily / Ubiquitin-activating enzyme E1, SCCH domain / Ubiquitin-activating enzyme E1, FCCH domain superfamily / Ubiquitin fold domain / Ubiquitin-activating enzyme E1 FCCH domain / Ubiquitin-activating enzyme E1 four-helix bundle / Ubiquitin-activating enzyme e1 C-terminal domain / Ubiquitin-activating enzyme, SCCH domain / Ubiquitin-activating enzyme, SCCH domain / Ubiquitin/SUMO-activating enzyme E1-like / Ubiquitin-activating enzyme E1, inactive adenylation domain, subdomain 1 / Ubiquitin-activating enzyme E1, Cys active site / Ubiquitin-activating enzyme active site. / ThiF/MoeB/HesA family / THIF-type NAD/FAD binding fold / ThiF family / Ubiquitin-activating enzyme / : / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme/RWD-like / Ubiquitin domain / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
ADENOSINE MONOPHOSPHATE / Ubiquitin/ISG15-conjugating enzyme E2 L6 / Ubiquitin-like protein ISG15 / Ubiquitin-like modifier-activating enzyme 7
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsWallace, I. / Kheewoong, B. / Prabu, J.R. / Vollrath, R. / von Gronau, S. / Schulman, B.A. / Swatek, K.N.
Funding support United Kingdom, Germany, European Union, 4items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UU_00018/10 United Kingdom
Royal SocietyRGS_R2_222185 United Kingdom
Max Planck Society Germany
European Research Council (ERC)European Union
CitationJournal: Nat Commun / Year: 2023
Title: Insights into the ISG15 transfer cascade by the UBE1L activating enzyme.
Authors: Iona Wallace / Kheewoong Baek / J Rajan Prabu / Ronnald Vollrath / Susanne von Gronau / Brenda A Schulman / Kirby N Swatek /
Abstract: The attachment of the ubiquitin-like protein ISG15 to substrates by specific E1-E2-E3 enzymes is a well-established signalling mechanism of the innate immune response. Here, we present a 3.45 Å ...The attachment of the ubiquitin-like protein ISG15 to substrates by specific E1-E2-E3 enzymes is a well-established signalling mechanism of the innate immune response. Here, we present a 3.45 Å cryo-EM structure of a chemically trapped UBE1L-UBE2L6 complex bound to activated ISG15. This structure reveals the details of the first steps of ISG15 recognition and UBE2L6 recruitment by UBE1L (also known as UBA7). Taking advantage of viral effector proteins from severe acute respiratory coronavirus 2 (SARS-CoV-2) and influenza B virus (IBV), we validate the structure and confirm the importance of the ISG15 C-terminal ubiquitin-like domain in the adenylation reaction. Moreover, biochemical characterization of the UBE1L-ISG15 and UBE1L-UBE2L6 interactions enables the design of ISG15 and UBE2L6 mutants with altered selectively for the ISG15 and ubiquitin conjugation pathways. Together, our study helps to define the molecular basis of these interactions and the specificity determinants that ensure the fidelity of ISG15 signalling during the antiviral response.
History
DepositionMar 22, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 13, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Ubiquitin-like modifier-activating enzyme 7
I: Ubiquitin-like protein ISG15
L: Ubiquitin/ISG15-conjugating enzyme E2 L6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)147,3744
Polymers147,0273
Non-polymers3471
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, In addition to gel filtration, the stoichiometry of the complex was confirmed by reducing the E1-E2 disulfide linkage and analysing the sample by SDS-PAGE.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4280 Å2
ΔGint-36 kcal/mol
Surface area43890 Å2

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Components

#1: Protein Ubiquitin-like modifier-activating enzyme 7 / Ubiquitin-activating enzyme 7 / D8 / Ubiquitin-activating enzyme E1 homolog


Mass: 111966.234 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBA7, UBE1L, UBE2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P41226
#2: Protein Ubiquitin-like protein ISG15 / Interferon-induced 15 kDa protein / Interferon-induced 17 kDa protein / IP17 / Ubiquitin cross- ...Interferon-induced 15 kDa protein / Interferon-induced 17 kDa protein / IP17 / Ubiquitin cross-reactive protein / hUCRP


Mass: 17147.637 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ISG15, G1P2, UCRP / Production host: Escherichia coli (E. coli) / References: UniProt: P05161
#3: Protein Ubiquitin/ISG15-conjugating enzyme E2 L6 / E2 ubiquitin-conjugating enzyme L6 / Retinoic acid-induced gene B protein / RIG-B / UbcH8 / ...E2 ubiquitin-conjugating enzyme L6 / Retinoic acid-induced gene B protein / RIG-B / UbcH8 / Ubiquitin carrier protein L6 / Ubiquitin-protein ligase L6


Mass: 17912.719 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBE2L6, UBCH8 / Production host: Escherichia coli (E. coli)
References: UniProt: O14933, E2 ubiquitin-conjugating enzyme
#4: Chemical ChemComp-AMP / ADENOSINE MONOPHOSPHATE


Mass: 347.221 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H14N5O7P / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP*YM
Has ligand of interestY
Has protein modificationY

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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
1Ternary complex consisting of UBE1L, UBE2L6, and full-length ISG15.COMPLEX#1-#30RECOMBINANT
2UBE2L6COMPLEX1RECOMBINANT
3ISG15COMPLEX1RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Trichoplusia ni (cabbage looper)7111
32Escherichia coli (E. coli)562
43Escherichia coli (E. coli)562
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 3581364 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0026753
ELECTRON MICROSCOPYf_angle_d0.5749287
ELECTRON MICROSCOPYf_dihedral_angle_d6.033990
ELECTRON MICROSCOPYf_chiral_restr0.0381135
ELECTRON MICROSCOPYf_plane_restr0.0071192

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