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- PDB-8seb: Cryo-EM structure of a single loaded human UBA7-UBE2L6-ISG15 aden... -

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Basic information

Entry
Database: PDB / ID: 8seb
TitleCryo-EM structure of a single loaded human UBA7-UBE2L6-ISG15 adenylate complex
Components
  • Ubiquitin-like modifier-activating enzyme 7
  • Ubiquitin-like protein ISG15
  • Ubiquitin/ISG15-conjugating enzyme E2 L6
KeywordsLIGASE / Signaling 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 / 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 / protein modification process / DDX58/IFIH1-mediated induction of interferon-alpha/beta / response to virus / PKR-mediated signaling / 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 / 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 / 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: 3.24 Å
AuthorsAfsar, M. / Jia, L. / Ruben, E.A. / Olsen, S.K.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Nat Commun / Year: 2023
Title: Cryo-EM structures of Uba7 reveal the molecular basis for ISG15 activation and E1-E2 thioester transfer.
Authors: Mohammad Afsar / GuanQun Liu / Lijia Jia / Eliza A Ruben / Digant Nayak / Zuberwasim Sayyad / Priscila Dos Santos Bury / Kristin E Cano / Anindita Nayak / Xiang Ru Zhao / Ankita Shukla / ...Authors: Mohammad Afsar / GuanQun Liu / Lijia Jia / Eliza A Ruben / Digant Nayak / Zuberwasim Sayyad / Priscila Dos Santos Bury / Kristin E Cano / Anindita Nayak / Xiang Ru Zhao / Ankita Shukla / Patrick Sung / Elizabeth V Wasmuth / Michaela U Gack / Shaun K Olsen /
Abstract: ISG15 plays a crucial role in the innate immune response and has been well-studied due to its antiviral activity and regulation of signal transduction, apoptosis, and autophagy. ISG15 is a ubiquitin- ...ISG15 plays a crucial role in the innate immune response and has been well-studied due to its antiviral activity and regulation of signal transduction, apoptosis, and autophagy. ISG15 is a ubiquitin-like protein that is activated by an E1 enzyme (Uba7) and transferred to a cognate E2 enzyme (UBE2L6) to form a UBE2L6-ISG15 intermediate that functions with E3 ligases that catalyze conjugation of ISG15 to target proteins. Despite its biological importance, the molecular basis by which Uba7 catalyzes ISG15 activation and transfer to UBE2L6 is unknown as there is no available structure of Uba7. Here, we present cryo-EM structures of human Uba7 in complex with UBE2L6, ISG15 adenylate, and ISG15 thioester intermediate that are poised for catalysis of Uba7-UBE2L6-ISG15 thioester transfer. Our structures reveal a unique overall architecture of the complex compared to structures from the ubiquitin conjugation pathway, particularly with respect to the location of ISG15 thioester intermediate. Our structures also illuminate the molecular basis for Uba7 activities and for its exquisite specificity for ISG15 and UBE2L6. Altogether, our structural, biochemical, and human cell-based data provide significant insights into the functions of Uba7, UBE2L6, and ISG15 in cells.
History
DepositionApr 8, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 11, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 23, 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
B: Ubiquitin-like protein ISG15
C: Ubiquitin/ISG15-conjugating enzyme E2 L6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)146,9604
Polymers146,6133
Non-polymers3471
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 111822.102 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 / Mutation: C78S
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: 17643.377 Da / Num. of mol.: 1 / Mutation: C98S,C102S,L121K
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

ComponentName: Cryo-EM structure of a single loaded human UBA7-UBE2L6-ISG15 adenylate complex
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
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: 3911 nm / Nominal defocus min: 100 nm
Image recordingElectron dose: 53 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.24 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 67077 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0029851
ELECTRON MICROSCOPYf_angle_d0.47913419
ELECTRON MICROSCOPYf_dihedral_angle_d12.8593719
ELECTRON MICROSCOPYf_chiral_restr0.0371496
ELECTRON MICROSCOPYf_plane_restr0.0041762

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