Browse
Submit Data
Databases
API
Help

Dataset Information

21 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Mechanistic insights into active site-associated polyubiquitination by the ubiquitin-conjugating enzyme Ube2g2.

ABSTRACT: Lys-48-linked polyubiquitination regulates a variety of cellular processes by targeting ubiquitinated proteins to the proteasome for degradation. Although polyubiquitination had been presumed to occur by transferring ubiquitin molecules, one at a time, from an E2 active site to a substrate, we recently showed that the endoplasmic reticulum-associated RING finger ubiquitin ligase gp78 can mediate the preassembly of Lys-48-linked polyubiquitin chains on the catalytic cysteine of its cognate E2 Ube2g2 and subsequent transfer to a substrate. Active site-linked polyubiquitin chains are detected in cells on Ube2g2 and its yeast homolog Ubc7p, but how these chains are assembled is unclear. Here, we show that gp78 forms an oligomer via 2 oligomerization sites, one of which is a hydrophobic segment located in the gp78 cytosolic domain. We further demonstrate that a gp78 oligomer can simultaneously associate with multiple Ube2g2 molecules. This interaction is mediated by a novel Ube2g2 surface distinct from the predicted RING binding site. Our data suggest that a large gp78-Ube2g2 heterooligomer brings multiple Ube2g2 molecules into close proximity, allowing ubiquitin moieties to be transferred between neighboring Ube2g2s to form active site-linked polyubiquitin chains.

SUBMITTER: Li W

PROVIDER: S-EPMC2644258 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Solution structure and dynamics of human ubiquitin conjugating enzyme Ube2g2.

Project description:Ube2g2 is an E2 enzyme which functions as part of the endoplasmic reticulum-associated degradation (ERAD) pathway responsible for identification and degradation of misfolded proteins in the endoplasmic reticulum. In tandem with a cognate E3 ligase, Ube2g2 assembles K48-linked polyubiquitin chains and then transfers them to substrate, leading ultimately to proteasomal degradation of the polyubiquitin-tagged substrate. We report here the solution structure and backbone dynamics of Ube2g2 solved by nuclear magnetic resonance spectroscopy. Although the solution structure agrees well with crystallographic structures for the E2 core, catalytically important loops (encompassing residues 95-107 and 130-135) flanking the active site cysteine are poorly defined. (15)N spin relaxation and residual dipolar coupling analysis directly demonstrates that these two loops are highly dynamic in solution. These results suggest that Ube2g2 requires one or more of its protein partners, such as cognate E3, acceptor ubiquitin substrate or thiolester-linked donor ubiquitin, to assume its catalytically relevant conformation. Within the NMR structural ensemble, interactions were observed between His94 and the highly mobile loop residues Asp98 and Asp99, supporting a possible role for His94 as a general base activated by the carboxylate side-chains of Asp98 or Asp99.

| S-EPMC2822102 | biostudies-literature

Structure of human ubiquitin-conjugating enzyme E2 G2 (UBE2G2/UBC7).

Project description:The human ubiquitin-conjugating enzyme E2 G2 (UBE2G2/UBC7) is involved in protein degradation, including a process known as endoplasmic reticulum-associated degradation (ERAD). The crystal structure of human UBE2G2/UBC7 was solved at 2.56 angstroms resolution. The UBE2G2 structure comprises a single domain consisting of an antiparallel beta-sheet with four strands, five alpha-helices and two 3(10)-helices. Structural comparison of human UBE2G2 with yeast Ubc7 indicated that the overall structures are similar except for the long loop region and the C-terminal helix. Superimposition of UBE2G2 on UbcH7 in a c-Cbl-UbcH7-ZAP70 ternary complex suggested that the two loop regions of UBE2G2 interact with the RING domain in a similar way to UbcH7. In addition, the extra loop region of UBE2G2 may interact with the RING domain or its neighbouring region and may be involved in the binding specificity and stability.

| S-EPMC2222581 | biostudies-literature

Conformational Dynamics Modulate Activation of the Ubiquitin Conjugating Enzyme Ube2g2.

Project description:The ubiquitin conjugating enzyme Ube2g2 together with its cognate E3 ligase gp78 catalyzes the synthesis of lysine-48 polyubiquitin chains constituting signals for the proteasomal degradation of misfolded proteins in the endoplasmic reticulum. Here, we employ NMR spectroscopy in combination with single-turnover diubiquitin formation assays to examine the role of the RING domain from gp78 in the catalytic activation of Ube2g2∼Ub conjugates. We find that approximately 60% of the Ube2g2∼Ub conjugates occupy a closed conformation in the absence of gp78-RING, with the population increasing to 82% upon gp78-RING binding. As expected, strong mutations in the hydrophobic patch residues of the ∼Ub moiety result in Ube2g2∼Ub populating only open states with corresponding loss of the ubiquitin conjugation activity. Less disruptive mutations introduced into the hydrophobic patch of the ∼Ub moiety also destabilize the closed conformational state, yet the corresponding effect on the ubiquitin conjugation activity ranges from complete loss to an enhancement of the catalytic activity. These results present a picture in which Ube2g2's active site is in a state of continual dynamic flux with the organization of the active site into a catalytically viable conformation constituting the rate-limiting step for a single ubiquitin ligation event. Ube2g2's function as a highly specific K48-polyubiquitin chain elongator leads us to speculate that this may be a strategy by which Ube2g2 reduces the probability of nonproductive catalytic outcomes in the absence of available substrate.

| S-EPMC5579538 | biostudies-literature

Mechanism of polyubiquitin chain recognition by the human ubiquitin conjugating enzyme Ube2g2.

Project description:Ube2g2 is a human ubiquitin conjugating (E2) enzyme involved in the endoplasmic reticulum-associated degradation pathway, which is responsible for the identification and degradation of unfolded and misfolded proteins in the endoplasmic reticulum compartment. The Ube2g2-specific role is the assembly of Lys-48-linked polyubiquitin chains, which constitutes a signal for proteasomal degradation when attached to a substrate protein. NMR chemical shift perturbation and paramagnetic relaxation enhancement approaches were employed to characterize the binding interaction between Ube2g2 and ubiquitin, Lys-48-linked diubiquitin, and Lys-63-linked diubiquitin. Results demonstrate that ubiquitin binds to Ube2g2 with an affinity of 90 ?M in two different orientations that are rotated by 180° in models generated by the RosettaDock modeling suite. The binding of Ube2g2 to Lys-48- and Lys-63-linked diubiquitin is primarily driven by interactions with individual ubiquitin subunits, with a clear preference for the subunit containing the free Lys-48 or Lys-63 side chain (i.e. the distal subunit). This preference is particularly striking in the case of Lys-48-linked diubiquitin, which exhibits an ?3-fold difference in affinities between the two ubiquitin subunits. This difference can be attributed to the partial steric occlusion of the subunit whose Lys-48 side chain is involved in the isopeptide linkage. As such, these results suggest that Lys-48-linked polyubiquitin chains may be designed to bind certain proteins like Ube2g2 such that the terminal ubiquitin subunit carrying the reactive Lys-48 side chain can be positioned properly for chain elongation regardless of chain length.

| S-EPMC3030398 | biostudies-literature

Design, synthesis, and anticancer activity evaluation of irreversible allosteric inhibitors of the ubiquitin-conjugating enzyme Ube2g2.

Project description:The RING finger-dependent ubiquitin ligase (E3) gp78, known as the tumor autocrine motility factor receptor, contributes to tumor progression. The protein interacts with its cognate ubiquitin-conjugating enzyme (E2), Ube2g2, via its RING domain and a unique region called G2BR that strongly binds to E2. The binding of G2BR to Ube2g2 allosterically enhances the binding of RING to E2, and the binding of RING triggers the departure of G2BR from E2 also in an allosteric fashion. Targeting these allosteric events, we developed a family of inhibitors that irreversibly block E2-E3 interactions and thereby eliminate the tumorigenic effect of gp78. One among 19 compounds screened with the NCI 60 tumor cell lines exhibited outstanding anticancer activities. At 10 ?M, it caused >50% growth inhibition to 40% of the cell lines; at 100 ?M, it showed lethiferous activity against most cell lines.

| S-EPMC6238722 | biostudies-literature

Mechanistic insight into the allosteric activation of a ubiquitin-conjugating enzyme by RING-type ubiquitin ligases.

Project description:Ubiquitin-conjugating enzymes (E2s) collaborate with the ubiquitin-activating enzyme (E1) and ubiquitin ligases (E3s) to attach ubiquitin to target proteins. RING-containing E3s simultaneously bind to E2s and substrates, bringing them into close proximity and thus facilitating ubiquitination. We show herein that, although the E3-binding site on the human E2 UbcH5b is distant from its active site, two RING-type minimal E3 modules lacking substrate-binding functions greatly stimulate the rate of ubiquitin release from the UbcH5b-ubiquitin thioester. Using statistical coupling analysis and mutagenesis, we identify and characterize clusters of coevolving and functionally linked residues within UbcH5b that span its E3-binding and active sites. Several UbcH5b mutants are defective in their stimulation by E3s despite their abilities to bind to these E3s, to form ubiquitin thioesters, and to release ubiquitin at a basal rate. One such mutation, I37A, is distant from both the active site and the E3-binding site of UbcH5b. Our studies reveal structural determinants for communication between distal functional sites of E2s and suggest that RING-type E3s activate E2s allosterically.

| S-EPMC1316884 | biostudies-literature

The E2 ubiquitin-conjugating enzymes direct polyubiquitination to preferred lysines.

Project description: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.

| S-EPMC2838281 | biostudies-literature

Molecular basis of Lys-63-linked polyubiquitination inhibition by the interaction between human deubiquitinating enzyme OTUB1 and ubiquitin-conjugating enzyme UBC13.

Project description:UBC13 is the only known E2 ubiquitin (Ub)-conjugating enzyme that produces Lys-63-linked Ub chain with its cofactor E2 variant UEV1a or MMS2. Lys-63-linked ubiquitination is crucial for recruitment of DNA repair and damage response molecules to sites of DNA double-strand breaks (DSBs). A deubiquitinating enzyme OTUB1 suppresses Lys-63-linked ubiquitination of chromatin surrounding DSBs by binding UBC13 to inhibit its E2 activity independently of the isopeptidase activity. OTUB1 strongly suppresses UBC13-dependent Lys-63-linked tri-Ub production, whereas it allows di-Ub production in vitro. The mechanism of this non-canonical OTUB1-mediated inhibition of ubiquitination remains to be elucidated. Furthermore, the atomic level information of the interaction between human OTUB1 and UBC13 has not been reported. Here, we determined the crystal structure of human OTUB1 in complex with human UBC13 and MMS2 at 3.15 Å resolution. The presented atomic-level interactions were confirmed by surface-plasmon resonance spectroscopy with structure-based mutagenesis. The designed OTUB1 mutants cannot inhibit Lys-63-linked Ub chain formation in vitro and histone ubiquitination and 53BP1 assembly around DSB sites in vivo. Finally, we propose a model for how capping of di-Ub by the OTUB1-UBC13-MMS2/UEV1a complex efficiently inhibits Lys-63-linked tri-Ub formation.

| S-EPMC3406671 | biostudies-literature

Ubiquitin in motion: structural studies of the ubiquitin-conjugating enzyme?ubiquitin conjugate.

Project description:Ubiquitination of proteins provides a powerful and versatile post-translational signal in the eukaryotic cell. The formation of a thioester bond between ubiquitin (Ub) and the active site of a ubiquitin-conjugating enzyme (E2) is critical for the transfer of Ub to substrates. Assembly of a functional ubiquitin ligase (E3) complex poised for Ub transfer involves recognition and binding of an E2?Ub conjugate. Therefore, full characterization of the structure and dynamics of E2?Ub conjugates is required for further mechanistic understanding of Ub transfer reactions. Here we present characterization of the dynamic behavior of E2?Ub conjugates of two human enzymes, UbcH5c?Ub and Ubc13?Ub, in solution as determined by nuclear magnetic resonance and small-angle X-ray scattering. Within each conjugate, Ub retains great flexibility with respect to the E2, indicative of highly dynamic species that adopt manifold orientations. The population distribution of Ub conformations is dictated by the identity of the E2: the UbcH5c?Ub conjugate populates an array of extended conformations, and the population of Ubc13?Ub conjugates favors a closed conformation in which the hydrophobic surface of Ub faces helix 2 of Ubc13. We propose that the varied conformations adopted by Ub represent available binding modes of the E2?Ub species and thus provide insight into the diverse E2?Ub protein interactome, particularly with regard to interaction with Ub ligases.

| S-EPMC3056393 | biostudies-literature

Structural insights into E1 recognition and the ubiquitin-conjugating activity of the E2 enzyme Cdc34.

Project description:Ubiquitin (Ub) signaling requires the sequential interactions and activities of three enzymes, E1, E2, and E3. Cdc34 is an E2 that plays a key role in regulating cell cycle progression and requires unique structural elements to function. The molecular basis by which Cdc34 engages its E1 and the structural mechanisms by which its unique C-terminal extension functions in Cdc34 activity are unknown. Here, we present crystal structures of Cdc34 alone and in complex with E1, and a Cdc34~Ub thioester mimetic that represents the product of Uba1-Cdc34 Ub transthiolation. These structures reveal conformational changes in Uba1 and Cdc34 and a unique binding mode that are required for transthiolation. The Cdc34~Ub structure reveals contacts between the Cdc34 C-terminal extension and Ub that stabilize Cdc34~Ub in a closed conformation and are critical for Ub discharge. Altogether, our structural, biochemical, and cell-based studies provide insights into the molecular mechanisms by which Cdc34 function in cells.

| S-EPMC6656757 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data