An NMR-based Model of the Ubiquitin-bound Human Ubiquitin Conjugation Complex Mms2·Ubc13

E2 conjugating enzymes form a thiol ester intermediate with ubiquitin, which is subsequently transferred to a substrate protein targeted for degradation. While all E2 proteins comprise a catalytic domain where the thiol ester is formed, several E2s (class II) have C-terminal extensions proposed to control substrate recognition, dimerization, or polyubiquitin chain formation. Here we present the novel solution structure of the class II E2 conjugating enzyme Ubc1 from Saccharomyces cerevisiae. The structure shows the N-terminal catalytic domain adopts an ␣/␤ fold typical of other E2 proteins. This domain is physically separated from its C-terminal domain by a 22-residue flexible tether. The C-terminal domain adopts a three-helix bundle that we have identified as an ubiquitin-associated domain (UBA). NMR chemical shift perturbation experiments show this UBA domain interacts in a regioselective manner with ubiquitin. This two-domain structure of Ubc1 was used to identify other UBA-containing class II E2 proteins, including human E2-25K, that likely have a similar architecture and to determine the role of the UBA domain in facilitating polyubiquitin chain formation.Labeling of proteins with the molecule ubiquitin is an important cellular function that is required for protein degradation, cell cycle control, stress response, DNA repair, signal transduction, transcriptional regulation, and vesicular traffic (1-4). In this process, the number and topology of the ubiquitin molecule chains underscores the fate of the substrate and determines the biochemical pathway followed. For example, labeling the substrate with a single ubiquitin (monoubiqutination) is important for cellular regulation (5, 6). On the other hand ubiquitin-dependent proteolysis, the process responsible for the turnover of damaged or misfolded proteins in the cell, involves labeling the substrate protein with a polyubiquitin chain that is subsequently recognized by the 26 S proteasome facilitating substrate degradation. The most common polyubiquitin chains are formed via isopeptide bond linkages between the C-terminal (Gly 76 ) of one ubiquitin molecule and the side chain ⑀-NH 2 from Lys 48 of another. However, other configurations are possible including the Lys 63 linkage, which are important in the postreplicative DNA repair pathway (7).The ubiquitination degradation pathway is described as a cascade of events in which ubiquitin is passed through three enzymes until it reaches a protein selected for degradation. The first step involves an ATP-dependent activation of ubiquitin by an ubiquitin-activating enzyme (E1) 1 forming a high energy E1-ubiquitin thiol ester complex. The activated ubiquitin is then passed from the E1 to an ubiquitin-conjugating enzyme (E2) forming a second thiol ester intermediate between the E2 and ubiquitin. Labeling the target protein with ubiquitin is catalyzed by an E3 ligase protein, either by direct transfer of the ubiquitin to the substrate from the E2 (RING E3) or by thiol ester formation between ubiquitin to an E3 (HECT E...

Section: The C Terminus Of Ubc1mentioning

confidence: 89%

mentioning

confidence: 99%

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

Merkley

Shaw

2004

“…With the yeast homolog of UbcH13/Uev1a, Ubc13/Mms2, this formation of Lys 63 linkages has been shown to be due to the noncovalent binding on Mms2 of the preceding Ub so as to transfer the Ub from the E2 specifically to Lys 63 (49,50). In this case, as in SCF working with Cdc34 (51), the E3 appears to accelerate on the substrate the same process of chain synthesis, which the E2 can slowly catalyze by itself.…”

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

386

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.

“…Accordingly, seven different topologies of polyubiquitin can be generated (excluding mixed topologies) (18). Although most ubiquitin-dependent proteasomal degradations were found to be mediated by Lys 48 polyubiquitin chains, certain evidence suggests that Lys 6 (19), Lys 11 (19,20), and Lys 29 (21) may also target substrates for proteasomal degradation. Polyubiquitinations through nonLys 48 lysines exist in vivo and play a role in a range of cellular processes (22,23).…”

mentioning

confidence: 99%

“…Because ubiquitin harbors seven lysine residues (Lys 6 , Lys 11 , Lys 27 , Lys 29 , Lys 33 , Lys 48 , and Lys 63 ), in principle, chains of polyubiquitin can be formed via a bond between the C-terminal glycine of one ubiquitin molecule and an ⑀-amine of any of the seven lysine residues of another ubiquitin. Accordingly, seven different topologies of polyubiquitin can be generated (excluding mixed topologies) (18).…”

mentioning

confidence: 99%

The E2 Ubiquitin-conjugating Enzymes Direct Polyubiquitination to Preferred Lysines

David

Ziv

Admon

et al. 2010

148

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).