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Query: UNIPROT:P62988 (Ubiquitin)
 4,326 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ubiquitin-mediated proteolysis utilizes a series of three key enzymes (E1, E2, and E3) to transfer and then covalently modify a substrate with ubiquitin. E2 conjugating enzymes are central proteins in this pathway responsible for the acceptance of a ubiquitin from the E1 enzyme and association with an E3 protein. All E2 enzymes covalently bind ubiquitin through a thiolester linkage between a conserved active-site cysteine on E2 and the C-terminal glycine on ubiquitin. It is not known whether E2 enzymes utilize similar surfaces and residues to coordinate a ubiquitin molecule and how this might contribute to any substrate specificity. In this work, we determined the structure of the human E2 enzyme UbcH8 (UBE2L6) covalently bound to ubiquitin by NMR spectroscopy. A disulfide bond mimicking the short-lived thiolester was formed between the two proteins providing a stable complex. Overall, the structure of UbcH8 does not undergo a significant conformational change upon forming a complex with ubiquitin. Chemical shift perturbation and cross-saturation experiments were used to identify contacts between UbcH8 and ubiquitin and those contacts used as inputs for HADDOCK molecular docking to produce the structure of the UbcH8-ubiquitin complex. An ensemble of 16 structures (root-mean-square deviation of 0.83 A) showed that ubiquitin interacts with the linker region prior to the alpha5 helix as well as residues near the catalytic site. This region corresponds to an area of negative potential on the UbcH8 surface and is considerably different from other E2-ubiquitin interaction sites. Our findings indicate the positioning of ubiquitin on UbcH8 would still allow interaction with E1 and E3 enzymes. Together, the results suggest the UbcH8-ubiquitin complex may provide an additional level of specificity in the ubiquitination pathway.
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PMID:The structure of the UbcH8-ubiquitin complex shows a unique ubiquitin interaction site. 1992 33

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.
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PMID:Solution structure and dynamics of human ubiquitin conjugating enzyme Ube2g2. 2001 27

The concept of cross-spectrum is applied in protein NMR spectroscopy to assist in the backbone sequential resonance assignment. Cross-spectrum analysis is used routinely to reveal correlations in frequency domains as a means to reveal common features contained in multiple time series. Here the cross-spectrum between related NMR spectra, for example HNCO and HN(CA)CO, can be calculated with point-by-point multiplications along their common C' carbon axis. In the resulting higher order cross-spectrum, an enhanced correlation signal occurs at every common i-1 carbon frequency allowing the amide proton H(N) (and nitrogen N) resonances from residues i and i-1 to be identified. The cross-spectrum approach is demonstrated using 2D spectra H(N)CO, H(NCA)CO, H(NCO)CACB, and H(N)CACB measured on a 15N/13C double-labeled Ubiquitin sample. These 2D spectra are used to calculate two pseudo-3D cross-spectra, H(i)-H(i)(-1)-C'(i)(-1) and H(i)-H(i)(-1)-CA(i)(-1)CB(i)(-1). We show using this approach, backbone resonances of H, C', CA, and CB can be fully assigned without ambiguity. The cross-spectrum principle is expected to offer an easy, practical, and more quantitative approach for heteronuclear backbone resonance assignment.
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PMID:A practical implementation of cross-spectrum in protein backbone resonance assignment. 2005 73

Cdc34 is an E2 ubiquitin-conjugating enzyme that functions in conjunction with SCF (Skp1.Cullin 1.F-box) E3 ubiquitin ligase to catalyze covalent attachment of polyubiquitin chains to a target protein. Here we identified direct interactions between the human Cdc34 C terminus and ubiquitin using NMR chemical shift perturbation assays. The ubiquitin binding activity was mapped to two separate Cdc34 C-terminal motifs (UBS1 and UBS2) that comprise residues 206-215 and 216-225, respectively. UBS1 and UBS2 bind to ubiquitin in the proximity of ubiquitin Lys(48) and C-terminal tail, both of which are key sites for conjugation. When bound to ubiquitin in one orientation, the Cdc34 UBS1 aromatic residues (Phe(206), Tyr(207), Tyr(210), and Tyr(211)) are probably positioned in the vicinity of ubiquitin C-terminal residue Val(70). Replacement of UBS1 aromatic residues by glycine or of ubiquitin Val(70) by alanine decreased UBS1-ubiquitin affinity interactions. UBS1 appeared to support the function of Cdc34 in vivo because human Cdc34(1-215) but not Cdc34(1-200) was able to complement the growth defect by yeast Cdc34 mutant strain. Finally, reconstituted IkappaBalpha ubiquitination analysis revealed a role for each adjacent pair of UBS1 aromatic residues (Phe(206)/Tyr(207), Tyr(210)/Tyr(211)) in conjugation, with Tyr(210) exhibiting the most pronounced catalytic function. Intriguingly, Cdc34 Tyr(210) was required for the transfer of the donor ubiquitin to a receptor lysine on either IkappaBalpha or a ubiquitin in a manner that depended on the neddylated RING sub-complex of the SCF. Taken together, our results identified a new ubiquitin binding activity within the human Cdc34 C terminus that contributes to SCF-dependent ubiquitination.
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PMID:The human Cdc34 carboxyl terminus contains a non-covalent ubiquitin binding activity that contributes to SCF-dependent ubiquitination. 2035 40

Ubiquitin is a versatile cellular signaling molecule that can form polymers of eight different linkages, and individual linkage types have been associated with distinct cellular functions. Though little is currently known about Lys11-linked ubiquitin chains, recent data indicate that they may be as abundant as Lys48 linkages and may be involved in vital cellular processes. Here we report the generation of Lys11-linked polyubiquitin in vitro, for which the Lys11-specific E2 enzyme UBE2S was fused to a ubiquitin binding domain. Crystallographic and NMR analyses of Lys11-linked diubiquitin reveal that Lys11-linked chains adopt compact conformations in which Ile44 is solvent exposed. Furthermore, we identify the OTU family deubiquitinase Cezanne as the first deubiquitinase with Lys11-linkage preference. Our data highlight the intrinsic specificity of the ubiquitin system that extends to Lys11-linked chains and emphasize that differentially linked polyubiquitin chains must be regarded as independent post-translational modifications.
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PMID:Lys11-linked ubiquitin chains adopt compact conformations and are preferentially hydrolyzed by the deubiquitinase Cezanne. 2062 74

Human E4B, also called UFD2a, is a U box-containing protein that functions as an E3 ubiquitin ligase and an E4 polyubiquitin chain elongation factor. E4B is thought to participate in the proteasomal degradation of misfolded or damaged proteins through association with chaperones. The U box domain is an anchor site for E2 ubiquitin-conjugating enzymes, but little is known of the binding mechanism. Using X-ray crystallography and NMR spectroscopy, we determined the structures of E4B U box free and bound to UbcH5c and Ubc4 E2s. Whereas previously characterized U box domains are homodimeric, we show that E4B U box is a monomer stabilized by a network of hydrogen bonds identified from scalar coupling measurements. These structural studies, complemented by calorimetry- and NMR-based binding assays, suggest an allosteric regulation of UbcH5c and Ubc4 by E4B U box and provide a molecular basis to understand how the ubiquitylation machinery involving E4B assembles.
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PMID:Molecular basis for the association of human E4B U box ubiquitin ligase with E2-conjugating enzymes UbcH5c and Ubc4. 2069 87

RAP80 plays a key role in signal transduction in the DNA damage response by recruiting proteins to DNA damage foci by binding K63-polyubiquitin chains with two tandem ubiquitin-interacting motifs (tUIM). It is generally recognized that the typically weak interaction between ubiquitin (Ub) and various recognition motifs is intensified by themes such as tandem recognition motifs and Ub polymerization to achieve biological relevance. However, it remains an intricate problem to develop a detailed molecular mechanism to describe the process that leads to amplification of the Ub signal. A battery of solution-state NMR methods and molecular dynamics simulations were used to demonstrate that RAP80-tUIM employs mono- and multivalent interactions with polyUb chains to achieve enhanced affinity in comparison to monoUb interactions for signal amplification. The enhanced affinity is balanced by unfavorable entropic effects that include partial quenching of rapid reorientation between individual UIM domains and individual Ub domains in the bound state. For the RAP80-tUIM-polyUb interaction, increases in affinity with increasing chain length are a result of increased numbers of mono- and multivalent binding sites in the longer polyUb chains. The mono- and multivalent interactions are characterized by intrinsically weak binding and fast off-rates; these weak interactions with fast kinetics may be an important factor underlying the transient nature of protein-protein interactions that comprise DNA damage foci.
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PMID:Mechanism for recognition of polyubiquitin chains: balancing affinity through interplay between multivalent binding and dynamics. 2069 91

Ataxin-3, the disease protein in the neurodegenerative disorder Spinocerebellar Ataxia Type 3 or Machado Joseph disease, is a cysteine protease implicated in the ubiquitin proteasome pathway. It contains multiple ubiquitin binding sites through which it anchors polyubiquitin chains of different linkages that are then cleaved by the N-terminal catalytic (Josephin) domain. The properties of the ubiquitin interacting motifs (UIMs) in the C-terminus of ataxin-3 are well established. Very little is known, however, about how two recently identified ubiquitin-binding sites in the Josephin domain contribute to ubiquitin chain binding and cleavage. In the current study, we sought to define the specific contribution of the Josephin domain to the catalytic properties of ataxin-3 and assess how the topology and affinity of these binding sites modulate ataxin-3 activity. Using NMR we modeled the structure of diUb/Josephin complexes and showed that linkage preferences are imposed by the topology of the two binding sites. Enzymatic studies further helped us to determine a precise hierarchy between the sites. We establish that the structure of Josephin dictates specificity for K48-linked chains. Site 1, which is close to the active site, is indispensable for cleavage. Our studies open the way to understand better the cellular function of ataxin-3 and its link to pathology.
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PMID:Understanding the role of the Josephin domain in the PolyUb binding and cleavage properties of ataxin-3. 2086 50

The emerging role of internal dynamics in protein fold and function requires new avenues of structure analysis. We analyzed the dynamically restrained conformational ensemble of ubiquitin generated from residual dipolar coupling data, in terms of protruding and buried atoms as well as interatomic distances, using four proximity-based algorithms, CX, DPX, PRIDE and PRIDE-NMR (http://hydra.icgeb.trieste.it/protein/). We found that Ubiquitin, this relatively rigid molecule has a highly diverse dynamic ensemble. The environment of protruding atoms is highly variable across conformers, on the other hand, only a part of buried atoms tends to fluctuate. The variability of the ensemble cautions against the use of single conformers when explaining functional phenomena. We also give a detailed evaluation of PRIDE-NMR on a wide dataset and discuss its usage in the light of the features of available NMR distance restraint sets in public databases.
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PMID:Probing dynamic protein ensembles with atomic proximity measures. 2088 64

Small Ubiquitin-like MOdifiers (SUMOs) are ubiquitin-like proteins known to covalently modify large number of cellular proteins. The mammalian SUMO family includes four paralogues, SUMO-1 through SUMO-4. Death-associated protein-6, Daxx, is a 740 residue important transcription corepressor known to represses transcriptional potential of several sumolyted transcription factors. Daxx also plays important role in apoptosis. Both terminals of Daxx harbor separate SUMO Interaction Motifs (SIM), which mediate its interaction with SUMO and hence the sumolyted transcription factors. The C-terminal SIM of Daxx preferentially binds SUMO-1. Practically complete (1)H, (13)C and (15)N resonance assignments for the complex between SUMO-1 and 20 residue Daxx C-terminal SIM peptide are reported here.
Biomol NMR Assign 2011 Apr
PMID:NMR chemical shift assignments of a complex between SUMO-1 and SIM peptide derived from the C-terminus of Daxx. 2092 12


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