Ubiquitin Manipulation by an E2 Conjugating Enzyme Using a Novel Covalent Intermediate

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 (Gly 76 ) 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 Gly 76 to Cys 76 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.The ubiquitin-dependent proteolysis pathway controls the removal of damaged and misfolded proteins in the cell. One of the key steps in this pathway is the assembly of a polyubiquitin chain that ultimately targets a substrate for degradation (1, 2). This process involves tagging of a substrate protein with an arrangement of four to eight ubiquitin molecules linked in series through the C-terminal Gly 76 of one ubiquitin molecule and the side-chain ⑀-NH 2 from Lys 48 of another (3). Once tagged, the polyubiquitinated protein is recognized by the 26 S proteasome, where it is degraded. The building of a polyubiquitin chain and subsequent labeling of a substrate protein is a complex process potentially involving the passage of ubiquitin through three different enzymes (1, 4, 5). Initially, ubiquitin is activated in an ATP-dependent step forming a high energy E1 1 -ubiquitin thioester complex. Ubiquitin is then transferred to an E2 or ubiquitin-conjugating enzyme forming a thioester intermediate. E2 proteins have been demonstrated recently to bind to the ubiquitin-like domain of the E1 providing insight into the mechanism in which the thioester-bound ubiquitin is passed to the E2 (6 -8). Two different E3 ligase proteins can catalyze the final passage of ubiquitin to the substrate. For RING E3 ligases, ubiquitin or a polyubiquitin chain is transferred directly from the E2 to the substrate, whereas HECT E3 ligases form a...

Polyubiquitination by HECT E3s and the Determinants of Chain Type Specificity

“…Based on previous genetic and biochemical interactions (18,29,32), we expected that only Ubc1, -4, and -5 would function with Rsp5. Ubc1 contains a C-terminal UBA ubiquitin binding domain that has been suggested to influence its polyubiquitination activities (30,37), so a Ubc1-⌬UBA protein was also analyzed. As shown in Fig.…”

Structural Basis of E2–25K/UBB+1 Interaction Leading to Proteasome Inhibition and Neurotoxicity

“…Rigid Interaction between the E2 and UBA Domains-Among E2 proteins, E2-25K and its yeast homolog, UBC1, are unique in that they contain a C-terminal UBA domain in addition to an E2 domain. The E2 and UBA domains of UBC1 are connected by a flexible, 22-residue linker and are presumably independent of one another (40,41). By contrast, the domains in E2-25K are linked by a short, six-residue tether and extensively interact via hydrophobic residues, resulting in a single globular topology.…”

Association of the Disordered C-terminus of CDC34 with a Catalytically Bound Ubiquitin

“…To identify if this occurs and to provide details of the interaction that might aid in developing a mechanism for CDC34 activity, we produced a CDC34-Ub thiolester mimetic using a stable disulfide linkage between the catalytic cysteine of CDC34 (C93) and Ub carrying a G76C substitution (Ub Cys ). This method has been successfully employed to study structures of other E2-Ub complexes including Ubc1-Ub 31 and, more recently, UbcH8-Ub. 32 Although these E2-Ub Cys complexes are catalytically inert, they have some benefits over the E2~Ub thiolester complexes, including longerterm stability at basic pH (N60 days) and the ability to purify the E2-Ub Cys complex after formation.…”

“…The observation that R48 from Ub Cys is involved in the interaction with the CDC34 catalytic domain is similar to results for Ubc1 and UbcH8. 31,32 To identify whether residues in the C-terminus of CDC34 had any interaction with Ub within the CDC34-Ub Cys complex, we performed similar experiments using the full-length CDC34 protein.…”