Degradation of misfolded and damaged proteins by the 26S proteasome requires the substrate to be tagged with a polyubiquitin chain. Assembly of polyubiquitin chains and subsequent substrate labeling potentially involves three enzymes, an E1, E2 and E3. E2 proteins are key enzymes and form a thiolester intermediate through their catalytic cysteine with the C-terminal glycine (Gly76) of ubiquitin. This thiolester intermediate is easily hydrolyzed in vitro and has eluded structural characterization. In order to overcome this, we have engineered a novel ubiquitin-E2 disulfide linked complex by mutating Gly76 to Cys76 in ubiquitin. Reaction of Ubc1, an E2 from S. cerevisiae, with this mutant ubiquitin resulted in an ubiquitin-E2 disulfide that could be purified and was stable for several weeks. Chemical shift perturbation analysis of the disulfide ubiquitin-Ubc1 complex by NMR spectroscopy reveals an ubiquitin-Ubc1 interface similar to that for the ubiquitin-E2 thiolester. In addition to the typical E2 catalytic domain, Ubc1 contains an UBA domain and we have utilized NMR spectroscopy to demonstrate that in this disulfide complex the UBA domain is freely accessible to non-covalently bind a second molecule of ubiquitin. The ability of the Ubc1 to bind two ubiquitin molecules suggests the UBA domain does not interact with the thiolester bound ubiquitin during polyubiquitin chain formation. Thus, construction of this novel ubiquitin-E2 disulfide provides a method to structurally characterize the first step in polyubiquitin chain assembly by Ubc1 and its related class II enzymes.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|