Abstract
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.
Highlights
The ubiquitin-dependent proteolysis pathway controls the removal of damaged and misfolded proteins in the cell
Structures of E2 proteins show that the catalytic domain has an ␣/-fold that is maintained upon complexation with either HECT [9] or RING [10, 11] E3 ligases
To circumvent this we have created a novel disulfide-linked ubiquitin-Ubc1 complex that mimics the ubiquitin-E2 thioester intermediate. We show that this ubiquitin-Ubc1 complex can be purified in high amounts, is stable for long periods of time, and has similar structural characteristics to the ubiquitin-E2 thioester intermediate. We have used this complex and NMR spectroscopy to show that the UBA domain can bind ubiquitin in a non-covalent fashion even in the presence of an ubiquitin molecule covalently bound at the catalytic domain
Summary
E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; Ubc150, S. cerevisiae Ubc (residues 1–150); Ub, S. cerevisiae K48R-ubiquitin; UbCys, K48R-ubiquitin with a G76C mutation; UBA, ubiquitin-associated; UbCys-Ubc, disulfide-linked complex between UbCys and Ubc (or Ubc150). To date the best details have been garnered from models derived from NMR chemical shift perturbation data for ubiquitin-E2 thioester intermediates [20] Attempts to stabilize this complex for more detailed structural and mechanistic experiments have met with limited success. To circumvent this we have created a novel disulfide-linked ubiquitin-Ubc complex that mimics the ubiquitin-E2 thioester intermediate. We show that this ubiquitin-Ubc complex can be purified in high amounts, is stable for long periods of time, and has similar structural characteristics to the ubiquitin-E2 thioester intermediate We have used this complex and NMR spectroscopy to show that the UBA domain can bind ubiquitin in a non-covalent fashion even in the presence of an ubiquitin molecule covalently bound at the catalytic domain.
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