Ubiquitin manipulation by an E2 conjugating enzyme using a novel covalent intermediate.
Degradation of misfolded and damaged proteins by the 26 S 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 thioester intermediate through their catalytic cysteine with the C-terminal glycine (Gly76) of ubiquitin. This thioester intermediate is easily hydrolyzed in vitro and has eluded structural characterization. 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 Saccharomyces 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 thioester. In addition to the typical E2 catalytic domain, Ubc1 contains an ubiquitin-associated (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 that the UBA domain does not interact with the thioester-bound ubiquitin during polyubiquitin chain formation. Thus, construction of this novel ubiquitin-E2 disulfide provides a method to characterize structurally the first step in polyubiquitin chain assembly by Ubc1 and its related class II enzymes.
Merkley
Nadine
N
Department of Biochemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada.
Barber
Kathryn R
KR
Shaw
Gary S
GS
D020134:Catalytic Domain; D009682:Magnetic Resonance Spectroscopy; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000502:physiology*; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000502:physiology*
D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C066752:UBC1 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes