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Structure paper

TitleStructural basis for transthiolation intermediates in the ubiquitin pathway.
Journal, issue, pagesNature, Vol. 633, Issue 8028, Page 216-223, Year 2024
Publish dateAug 14, 2024
AuthorsTomasz Kochańczyk / Zachary S Hann / Michaelyn C Lux / Avelyn Mae V Delos Reyes / Cheng Ji / Derek S Tan / Christopher D Lima /
PubMed AbstractTransthiolation (also known as transthioesterification) reactions are used in the biosynthesis of acetyl coenzyme A, fatty acids and polyketides, and for post-translational modification by ubiquitin ...Transthiolation (also known as transthioesterification) reactions are used in the biosynthesis of acetyl coenzyme A, fatty acids and polyketides, and for post-translational modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins. For the Ub pathway, E1 enzymes catalyse transthiolation from an E1~Ub thioester to an E2~Ub thioester. Transthiolation is also required for transfer of Ub from an E2~Ub thioester to HECT (homologous to E6AP C terminus) and RBR (ring-between-ring) E3 ligases to form E3~Ub thioesters. How isoenergetic transfer of thioester bonds is driven forward by enzymes in the Ub pathway remains unclear. Here we isolate mimics of transient transthiolation intermediates for E1-Ub(T)-E2 and E2-Ub(T)-E3 complexes (where T denotes Ub in a thioester or Ub undergoing transthiolation) using a chemical strategy with native enzymes and near-native Ub to capture and visualize a continuum of structures determined by single-particle cryo-electron microscopy. These structures and accompanying biochemical experiments illuminate conformational changes in Ub, E1, E2 and E3 that are coordinated with the chemical reactions to facilitate directional transfer of Ub from each enzyme to the next.
External linksNature / PubMed:39143218 / PubMed Central
MethodsEM (single particle)
Resolution2.5 - 4.16 Å
Structure data

EMDB-44200, PDB-9b55:
Ubiquitin E2-Ub-E3 HECT tetrahedral transthiolation intermediate mimic - state 1
Method: EM (single particle) / Resolution: 3.23 Å

EMDB-44201, PDB-9b56:
Ubiquitin E2-Ub-E3 HECT tetrahedral transthiolation intermediate mimic - state 2
Method: EM (single particle) / Resolution: 3.35 Å

EMDB-44202, PDB-9b57:
Ubiquitin E2-Ub-E3 HECT tetrahedral transthiolation intermediate mimic - state 3
Method: EM (single particle) / Resolution: 3.37 Å

EMDB-44203, PDB-9b58:
Ubiquitin E2-Ub-E3 HECT tetrahedral transthiolation intermediate mimic - state 4
Method: EM (single particle) / Resolution: 3.39 Å

EMDB-44204, PDB-9b59:
Ubiquitin E2-Ub-E3 HECT tetrahedral transthiolation intermediate mimic - state 5
Method: EM (single particle) / Resolution: 3.49 Å

EMDB-44205, PDB-9b5a:
Ubiquitin E2-Ub-E3 HECT tetrahedral transthiolation intermediate mimic - state 6
Method: EM (single particle) / Resolution: 3.65 Å

EMDB-44206, PDB-9b5b:
Ubiquitin E2-Ub-E3 HECT tetrahedral transthiolation intermediate mimic - state 7
Method: EM (single particle) / Resolution: 3.31 Å

EMDB-44207, PDB-9b5c:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (doubly Ub-loaded) - consensus map and model
Method: EM (single particle) / Resolution: 2.5 Å

EMDB-44208, PDB-9b5d:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (doubly Ub-loaded) - Ub(T) class 1 map and model from consensus
Method: EM (single particle) / Resolution: 2.8 Å

EMDB-44209, PDB-9b5e:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (doubly Ub-loaded) - Ub(T) class 10 map and model from consensus
Method: EM (single particle) / Resolution: 2.81 Å

EMDB-44210, PDB-9b5f:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (doubly Ub-loaded) - cluster 1 map and model (Ub(A)/ATP/Mg)
Method: EM (single particle) / Resolution: 2.78 Å

EMDB-44211, PDB-9b5g:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (doubly Ub-loaded) - cluster 2 map and model (Ub(A)/ATP/Mg)
Method: EM (single particle) / Resolution: 2.67 Å

EMDB-44212, PDB-9b5h:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (doubly Ub-loaded) - cluster 3 map and model (Ub(A)-AMP/PPi/Mg)
Method: EM (single particle) / Resolution: 2.69 Å

EMDB-44213, PDB-9b5i:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (doubly Ub-loaded) - cluster 4 map and model (Ub(A)-AMP/PPi/Mg)
Method: EM (single particle) / Resolution: 2.7 Å

EMDB-44214, PDB-9b5j:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (doubly Ub-loaded) - cluster 5 map and model (Ub(A)-AMP)
Method: EM (single particle) / Resolution: 2.86 Å

EMDB-44215, PDB-9b5k:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (doubly Ub-loaded) - Ub(T) class 1 map and model from cluster 1 (Ub(A)/ATP/Mg)
Method: EM (single particle) / Resolution: 3.16 Å

EMDB-44216, PDB-9b5l:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (doubly Ub-loaded) - Ub(T) class 10 map and model from cluster 5 (Ub(A)-AMP)
Method: EM (single particle) / Resolution: 3.3 Å

EMDB-44217, PDB-9b5m:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (singly Ub-loaded) - consensus map and model
Method: EM (single particle) / Resolution: 2.79 Å

EMDB-44218, PDB-9b5n:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (singly Ub-loaded) - Ub(T) class 1 map and model from consensus
Method: EM (single particle) / Resolution: 3.12 Å

EMDB-44219, PDB-9b5o:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (singly Ub-loaded) - Ub(T) class 10 map and model from consensus
Method: EM (single particle) / Resolution: 3.19 Å

EMDB-44220, PDB-9b5p:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (singly Ub-loaded) - cluster 1 map and model (ATP/Mg)
Method: EM (single particle) / Resolution: 3.08 Å

EMDB-44221, PDB-9b5q:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (singly Ub-loaded) - cluster 2 map and model (ATP/Mg)
Method: EM (single particle) / Resolution: 2.95 Å

EMDB-44222, PDB-9b5r:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (singly Ub-loaded) - cluster 3 map and model (ATP/Mg)
Method: EM (single particle) / Resolution: 2.95 Å

EMDB-44223, PDB-9b5s:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (singly Ub-loaded) - cluster 4 map and model (ATP/Mg)
Method: EM (single particle) / Resolution: 2.96 Å

EMDB-44224, PDB-9b5t:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (singly Ub-loaded) - cluster 5 map and model (ATP/Mg)
Method: EM (single particle) / Resolution: 3.16 Å

EMDB-44225, PDB-9b5u:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (singly Ub-loaded) - Ub(T) class 1 map and model from cluster 1 (ATP/Mg)
Method: EM (single particle) / Resolution: 3.67 Å

EMDB-44226, PDB-9b5v:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (singly Ub-loaded) - Ub(T) class 10 map and model from cluster 1 (ATP/Mg)
Method: EM (single particle) / Resolution: 3.94 Å

EMDB-44227, PDB-9b5w:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (singly Ub-loaded) - Ub(T) class 1 map and model from cluster 5 (ATP/Mg)
Method: EM (single particle) / Resolution: 3.96 Å

EMDB-44228, PDB-9b5x:
Ubiquitin E1-Ub-E2 tetrahedral transthiolation intermediate mimic (singly Ub-loaded) - Ub(T) class 10 map and model from cluster 5 (ATP/Mg)
Method: EM (single particle) / Resolution: 4.16 Å

Chemicals

PDB-1aiv:
APO OVOTRANSFERRIN

ChemComp-MG:
Unknown entry

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

ChemComp-POP:
PYROPHOSPHATE 2-

ChemComp-AMP:
ADENOSINE MONOPHOSPHATE / AMP*YM

Source
  • schizosaccharomyces pombe 972h- (yeast)
KeywordsLIGASE / UBIQUITIN / E2 / E3 / HECT / NEDD4 / RSP5 / PUB2 / UBC4 / TRANSTHIOESTERIFICATION / THIOESTER / TRANSTHIOLATION / TETRAHEDRAL INTERMEDIATE / ADENYLATION / INHIBITOR / NUCLEUS / PHOSPHOPROTEIN / UBL CONJUGATION PATHWAY / UBL / ATP ATP-BINDING / AMP / NUCLEOTIDE-BINDING / ISOPEPTIDE BOND / E1 / UBA1 / ATP / ATP-binding

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