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- EMDB-16891: Structure of the UBE1L activating enzyme bound to ISG15 and UBE2L6 -

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Basic information

Entry
Database: EMDB / ID: EMD-16891
TitleStructure of the UBE1L activating enzyme bound to ISG15 and UBE2L6
Map data
Sample
  • Complex: Ternary complex consisting of UBE1L, UBE2L6, and full-length ISG15.
    • Complex: UBE2L6
    • Complex: ISG15
    • Protein or peptide: Ubiquitin-like modifier-activating enzyme 7
    • Protein or peptide: Ubiquitin-like protein ISG15
    • Protein or peptide: Ubiquitin/ISG15-conjugating enzyme E2 L6
  • Ligand: ADENOSINE MONOPHOSPHATE
Keywordsubiquitin-like / ISG15 / interferon-stimulated genes / antiviral / ANTIVIRAL PROTEIN
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 / E2 ubiquitin-conjugating enzyme ...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 / E2 ubiquitin-conjugating enzyme / negative regulation of type I interferon-mediated signaling pathway / negative regulation of viral genome replication / 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 / Negative regulators of DDX58/IFIH1 signaling / integrin-mediated signaling pathway / Termination of translesion DNA synthesis / response to virus / protein modification process / DDX58/IFIH1-mediated induction of interferon-alpha/beta / modification-dependent protein catabolic process / PKR-mediated signaling / ISG15 antiviral mechanism / protein tag activity / protein polyubiquitination / ubiquitin-protein transferase activity / Interferon alpha/beta signaling / positive regulation of type II interferon production / Antigen processing: Ubiquitination & Proteasome degradation / integrin binding / ubiquitin-dependent protein catabolic process / defense response to virus / protein ubiquitination / defense response to bacterium / Amyloid fiber formation / innate immune response / DNA damage response / ubiquitin protein ligase binding / 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 four-helix bundle ...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 / Ubiquitin-activating enzyme / THIF-type NAD/FAD binding fold / ThiF family / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / Ubiquitin-conjugating enzyme/RWD-like / Ubiquitin domain / Ubiquitin family / Ubiquitin homologues / Ubiquitin-like domain / Ubiquitin domain profile. / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
Ubiquitin/ISG15-conjugating enzyme E2 L6 / Ubiquitin-like protein ISG15 / Ubiquitin-like modifier-activating enzyme 7
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsWallace I / Kheewoong B / Prabu JR / Vollrath R / von Gronau S / Schulman BA / Swatek KN
Funding support United Kingdom, Germany, European Union, 4 items
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, 2023-
Header (metadata) releaseDec 13, 2023-
Map releaseDec 13, 2023-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16891.png

Downloads & links

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Map

FileDownload / File: emd_16891.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 288 pix.
= 245.146 Å		0.85 Å/pix.
x 288 pix.
= 245.146 Å		0.85 Å/pix.
x 288 pix.
= 245.146 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8512 Å
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Contour LevelBy AUTHOR: 0.024
Minimum - Maximum-0.050229788 - 0.10205519
Average (Standard dev.)0.00028231138 (±0.0026009008)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 245.1456 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16891_msk_1.map
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Additional map: #1

Fileemd_16891_additional_1.map
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Half map: #2

Fileemd_16891_half_map_1.map
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Half map: #1

Fileemd_16891_half_map_2.map
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Sample components

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Entire : Ternary complex consisting of UBE1L, UBE2L6, and full-length ISG15.

EntireName: Ternary complex consisting of UBE1L, UBE2L6, and full-length ISG15.
Components
  • Complex: Ternary complex consisting of UBE1L, UBE2L6, and full-length ISG15.
    • Complex: UBE2L6
    • Complex: ISG15
    • Protein or peptide: Ubiquitin-like modifier-activating enzyme 7
    • Protein or peptide: Ubiquitin-like protein ISG15
    • Protein or peptide: Ubiquitin/ISG15-conjugating enzyme E2 L6
  • Ligand: ADENOSINE MONOPHOSPHATE

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Supramolecule #1: Ternary complex consisting of UBE1L, UBE2L6, and full-length ISG15.

SupramoleculeName: Ternary complex consisting of UBE1L, UBE2L6, and full-length ISG15.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: UBE2L6

SupramoleculeName: UBE2L6 / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: ISG15

SupramoleculeName: ISG15 / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Ubiquitin-like modifier-activating enzyme 7

MacromoleculeName: Ubiquitin-like modifier-activating enzyme 7 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 111.966234 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GSMDALDASK LLDEELYSRQ LYVLGSPAMQ RIQGARVLVS GLQGLGAEVA KNLVLMGVGS LTLHDPHPTC WSDLAAQFLL SEQDLERSR AEASQELLAQ LNRAVQVVVH TGDITEDLLL DFQVVVLTAA KLEEQLKVGT LCHKHGVCFL AADTRGLVGQ L FCDFGEDF ...String:
GSMDALDASK LLDEELYSRQ LYVLGSPAMQ RIQGARVLVS GLQGLGAEVA KNLVLMGVGS LTLHDPHPTC WSDLAAQFLL SEQDLERSR AEASQELLAQ LNRAVQVVVH TGDITEDLLL DFQVVVLTAA KLEEQLKVGT LCHKHGVCFL AADTRGLVGQ L FCDFGEDF TVQDPTEAEP LTAAIQHISQ GSPGILTLRK GANTHYFRDG DLVTFSGIEG MVELNDCDPR SIHVREDGSL EI GDTTTFS RYLRGGAITE VKRPKTVRHK SLDTALLQPH VVAQSSQEVH HAHCLHQAFC ALHKFQHLHG RPPQPWDPVD AET VVGLAR DLEPLKRTEE EPLEEPLDEA LVRTVALSSA GVLSPMVAML GAVAAQEVLK AISRKFMPLD QWLYFDALDC LPED GELLP SPEDCALRGS RYDGQIAVFG AGFQEKLRRQ HYLLVGAGAI GCELLKVFAL VGLGAGNSGG LTVVDMDHIE RSNLS RQFL FRSQDVGRPK AEVAAAAARG LNPDLQVIPL TYPLDPTTEH IYGDNFFSRV DGVAAALDSF QARRYVAARC THYLKP LLE AGTSGTWGSA TVFMPHVTEA YRAPASAAAS EDAPYPVCTV RYFPSTAEHT LQWARHEFEE LFRLSAETIN HHQQAHT SL ADMDEPQTLT LLKPVLGVLR VRPQNWQDCV AWALGHWKLC FHYGIKQLLR HFPPNKVLED GTPFWSGPKQ CPQPLEFD T NQDTHLLYVL AAANLYAQMH GLPGSQDWTA LRELLKLLPQ PDPQQMAPIF ASNLELASAS AEFGPEQQKE LNKALEVWS VGPPLKPLMF EKDDDSNFHV DFVVAAASLR CQNYGIPPVN RAQSKRIVGQ IIPAIATTTA AVAGLLGLEL YKVVSGPRPR SAFRHSYLH LAENYLIRYM PFAPAIQTFH HLKWTSWDRL KVPAGQPERT LESLLAHLQE QHGLRVRILL HGSALLYAAG W SPEKQAQH LPLRVTELVQ QLTGQAPAPG QRVLVLELSC EGDDEDTAFP PLHYEL

UniProtKB: Ubiquitin-like modifier-activating enzyme 7

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Macromolecule #2: Ubiquitin-like protein ISG15

MacromoleculeName: Ubiquitin-like protein ISG15 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 17.147637 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MGWDLTVKML AGNEFQVSLS SSMSVSELKA QITQKIGVHA FQQRLAVHPS GVALQDRVPL ASQGLGPGST VLLVVDKSDE PLSILVRNN KGRSSTYEVR LTQTVAHLKQ QVSGLEGVQD DLFWLTFEGK PLEDQLPLGE YGLKPLSTVF MNLRLRGG

UniProtKB: Ubiquitin-like protein ISG15

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Macromolecule #3: Ubiquitin/ISG15-conjugating enzyme E2 L6

MacromoleculeName: Ubiquitin/ISG15-conjugating enzyme E2 L6 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: E2 ubiquitin-conjugating enzyme
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 17.912719 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GPMMASMRVV KELEDLQKKP PPYLRNLSSD DANVLVWHAL LLPDQPPYHL KAFNLRISFP PEYPFKPPMI KFTTKIYHPN VDENGQICL PIISSENWKP STKTSQVLEA LNVLVNRPNI REPLRMDLAD LLTQNPELFR KNAEEFTLRF GVDRPS

UniProtKB: Ubiquitin/ISG15-conjugating enzyme E2 L6

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Macromolecule #4: ADENOSINE MONOPHOSPHATE

MacromoleculeName: ADENOSINE MONOPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: AMP
Molecular weightTheoretical: 347.221 Da
Chemical component information

ChemComp-AMP:
ADENOSINE MONOPHOSPHATE / AMP*YM / Adenosine monophosphate

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 3581364
FSC plot (resolution estimation)

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