modeling of the cryptic E2∼ubiquitin-binding site of E6-associated protein (E6AP)/UBE3A reveals the mechanism of polyubiquitin chain assembly.
Journal:
The Journal of biological chemistry
PMID:
28924046
Abstract
To understand the mechanism for assembly of Lys-linked polyubiquitin degradation signals, we previously demonstrated that the E6AP/UBE3A ligase harbors two functionally distinct E2∼ubiquitin-binding sites: a high-affinity Site 1 required for E6AP Cys∼ubiquitin thioester formation and a canonical Site 2 responsible for subsequent chain elongation. Ordered binding to Sites 1 and 2 is here revealed by observation of UbcH7∼ubiquitin-dependent substrate inhibition of chain formation at micromolar concentrations. To understand substrate inhibition, we exploited the PatchDock algorithm to model UbcH7∼ubiquitin bound to Site 1, validated by chain assembly kinetics of selected point mutants. The predicted structure buries an extensive solvent-excluded surface bringing the UbcH7∼ubiquitin thioester bond within 6 Å of the Cys nucleophile. Modeling onto the active E6AP trimer suggests that substrate inhibition arises from steric hindrance between Sites 1 and 2 of adjacent subunits. Confirmation that Sites 1 and 2 function in was demonstrated by examining the effect of E6APC820A on wild-type activity and single-turnover pulse-chase kinetics. A cyclic proximal indexation model proposes that Sites 1 and 2 function in tandem to assemble thioester-linked polyubiquitin chains from the proximal end attached to Cys before stochastic transfer to the target protein. Non-reducing SDS-PAGE confirms assembly of the predicted Cys-linked I-polyubiquitin thioester intermediate. Other studies suggest that Glu serves as a general base to generate the Cys thiolate within the low dielectric binding interface and Arg functions to orient Glu and to stabilize the incipient anionic transition state during thioester exchange.
Authors
Keywords
Amino Acid Substitution
Animals
Binding Sites
Biocatalysis
Cattle
Computational Biology
Cysteine
Expert Systems
Humans
Iodine Radioisotopes
Isoenzymes
Models, Molecular
Molecular Docking Simulation
Mutagenesis, Site-Directed
Point Mutation
Protein Conformation
Protein Multimerization
Recombinant Fusion Proteins
Ubiquitin
Ubiquitin-Conjugating Enzymes
Ubiquitin-Protein Ligases
Ubiquitination