Solution Structure of the Flexible Class II Ubiquitin-conjugating Enzyme Ubc1 Provides Insights for Polyubiquitin Chain Assembly

Self Cite

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...

“…1) ) is present. Unlike the catalytic domain, the UBA domain is able to interact with ubiquitin in a non-covalent manner (15).…”

Section: Resultsmentioning

confidence: 99%

Section: Ubc1 Coordinates Two Ubiquitin Molecules At Differentmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Merkley

Barber

Shaw

2005

Self Cite

“…S4A, left), whereas in the single arginine experiments, this E2 enzyme utilized all single arginine derivatives, excluding the Arg 48 ubiquitin (supplemental Fig. S4A, right), as reported previously (32). The mammalian ortholog of the yeast Ubc13-FIGURE 1.…”

Section: E2 Enzymes Autoubiquitinate Both Their Active Site Cysteine mentioning

confidence: 99%

The E2 Ubiquitin-conjugating Enzymes Direct Polyubiquitination to Preferred Lysines

David

Ziv

Admon

et al. 2010

142

125

The ubiquitin-proteasome pathway plays a crucial role in many cellular processes by degrading substrates tagged by polyubiquitin chains, linked mostly through lysine 48 of ubiquitin. Although polymerization of ubiquitin via its six other lysine residues exists in vivo as part of various physiological pathways, the molecular mechanisms that determine the type of polyubiquitin chains remained largely unknown. We undertook a systematic, in vitro, approach to evaluate the role of E2 enzymes in determining the topology of polyubiquitin. Because this study was performed in the absence of an E3 enzyme, our data indicate that the E2 enzymes are capable of directing the ubiquitination process to distinct subsets of ubiquitin lysines, depending on the specific E2 utilized. Moreover, our findings are in complete agreement with prior analyses of lysine preference assigned to certain E2s in the context of E3 (in vitro and in vivo). Finally, our findings support the rising notion that the functional unit of E2 is a dimer. To our knowledge, this is the first systematic indication for the involvement of E2 enzymes in specifying polyubiquitin chain assembly.In eukaryotic cells, most proteins are degraded by the 26 S proteasome, which hydrolyzes in an ATP-dependant manner, both ubiquitin-conjugated and certain non-ubiquitinated proteins. In addition to its role in the turnover of damaged or misfolded proteins, the proteasome controls the cell cycle and other processes through the degradation of critical regulatory components and transcription factors (1-3). Upon association of ubiquitinated targets with the proteasome, ubiquitin molecules are proteolytically removed for reuse, whereas the unfolded substrates are fed into the 20 S catalytic core, where they are digested into small peptides (4, 5).Protein ubiquitination is a multistep process orchestrated by the concerted action of three enzymes. The chain reaction begins with a ubiquitin-activating enzyme (E1), which initially adenylates the C-terminal glycine of ubiquitin. Next, a thioester bond is formed between the activated C terminus of ubiquitin and a cysteine residue of the E1. A ubiquitin-conjugating enzyme (E2) acquires the activated ubiquitin through a transthioesterification reaction. Finally, a RING ubiquitin-protein ligase (E3) recruits the substrate and guides the transfer of the ubiquitin from the E2 active site cysteine to the substrate. An ⑀-amine of a lysine residue on the substrate (or of additional ubiquitin) attacks the thioester bond between the ubiquitin and the E2 enzyme, forming an isopeptide bond with the C-terminal glycine of the ubiquitin (6 -8). Alternatively, when a HECT E3 catalyzes the transfer of the ubiquitin from the E2 to the target, an intermediate complex, of the activated ubiquitin and the active site cysteine of the HECT domain E3, is formed (9).

“…4). It has been reported that the C-terminal UBA domain of UbcH1 is required for polyUb chain synthesis (31,52), and if their UBA domain is deleted, UbcH1 forms irregular Ub chains (53). Presumably, this Ubbinding domain orients the Ub and determines the type of isopeptide linkage formed (in a similar fashion as Mms2 with Ubc13 (49)).…”

Section: Formation Of Homogeneous or Heterogeneous Forked Ubmentioning

confidence: 99%

Certain Pairs of Ubiquitin-conjugating Enzymes (E2s) and Ubiquitin-Protein Ligases (E3s) Synthesize Nondegradable Forked Ubiquitin Chains Containing All Possible Isopeptide Linkages

Kim

Lledı́as

et al. 2007

382

407

It is generally assumed that a specific ubiquitin ligase (E3)linksIn eukaryotic cells, ubiquitination serves to target regulatory and misfolded proteins for rapid degradation by proteasomes (1-3), to trigger endocytosis of membrane proteins (4), and also to allow specific protein-protein associations important in signal transduction, DNA repair, and gene transcription (5-8). Protein ubiquitination involves formation of isopeptide linkages between the C-terminal carboxyl group of a ubiquitin (Ub) 4 and an ⑀-amino group on a lysine on the protein substrate or a preceding Ub to form a polyUb chain. To synthesize such linkages, the C-terminal carboxyl group of a Ub is first activated by formation of a thioester bond with a cysteine on the Ubactivating enzyme (E1), and the activated Ub is then transferred as a thioester to one of the 20 -40 Ub-conjugating enzymes (E2) of the cell. The formation of a Ub chain on the substrate is then catalyzed by a Ub ligase (E3), which binds the substrate and an E2. Several families of E3s exist that differ in structure and mechanism. If ubiquitination is catalyzed by a member of the Ring finger or the U-box E3 family, the activated Ub is transferred from the E2 directly to a lysine on the protein substrate or to a preceding Ub. The abundant Ring finger and the related U-box families are small monomeric proteins that bind the substrate at one end and then in that vicinity release the reactive Ub from the E2-Ub thioester (9, 10). If ubiquitination is catalyzed by an E3 of the HECT domain family, the activated Ub is transferred from the E2 first to a cysteine on the E3 to form another thioester bond and then to the substrate or to a preceding Ub * This work was supported by grants from the NIGMS, the High Q Foundation, the Fund for Innovation from Elan Corp. (to A. L. G.), the National Institutes of Health (to S. P. G.), and National Institutes of Health R01 Grant GM65267 (to D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 The abbreviations used are: Ub, ubiquitin; UPKn, ubiquitin peptide modified at lysine n by ubiquitination; Forked chain, Ub chain in which two Ub chains are linked to the adjacent lysines on the preceding Ub; E1, Ubactivating enzyme; E2, Ub-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase; LC-MSMS liquid chromatography-tandem mass spectrometry; SIM, selective ion monitoring.