Characterization of the binding interface between ubiquitin and class I human ubiquitin-conjugating enzyme 2b by multidimensional heteronuclear NMR spectroscopy in solution 1 1Edited by P. E. Wright

Miura, Takaaki; Klaus, Werner; Gsell, Bernard; Miyamoto, Chikara; Senn, Hans

doi:10.1006/jmbi.1999.2859

Cited by 92 publications

(74 citation statements)

References 48 publications

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“…The binding area of ubiquitin for E2 is shown to overlap considerably with that for E1 by NMR studies. 36 The binding area of Atg12 for Atg7 and Atg10 might also overlap and share the same hydrophobic patch.…”

Section: Resultsmentioning

confidence: 99%

The Crystal Structure of Plant ATG12 and its Biological Implication in Autophagy

Suzuki

Yoshimoto²,

Fujioka³

et al. 2005

Autophagy

111

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Previously published online as a Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/abstract.php?id=1859 KEY WORDSAtATG12, autophagy, crystal structure, posttranslational modifier, ubiquitin-like protein ABBREVIATIONS AtArabidopsis thaliana PE phosphatidylethanolamine PAS pre-autophagosomal structure GST glutathione S-transferase MIRAS multiple isomorphous replacement with anomalous scattering ACKNOWLEDGEMENTSWe thank Dr. T. Hanada for valuable comments and suggestions on the manuscript. This work was supported by Grant-in-Aids for Scientific Research on Priority Areas and National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan. This work was carried out under the NIBB Cooperative Research Program (4-148). Research PaperThe Crystal Structure of Plant ATG12 and its Biological Implication in Autophagy ABSTRACTAtg12 is a post-translational modifier that is activated and conjugated to its single target, Atg5, by a ubiquitin-like conjugation system. The Atg12-Atg5 conjugate is essential for autophagy, the bulk degradation process of cytoplasmic components by the vacuolar/ lysosomal system. Here, we demonstrate that the Atg12 conjugation system exists in Arabidopsis and is essential for plant autophagy as well as in yeast and mammals. We also report the crystal structure of Arabidopsis thaliana (At) ATG12 at 1.8 Å resolution. Despite no obvious sequence homology with ubiquitin, the structure of AtATG12 shows a ubiquitin fold strikingly similar to those of mammalian homologs of Atg8, the other ubiquitinlike modifier essential for autophagy, which is conjugated to phosphatidylethanolamine. Two types of hydrophobic patches are present on the surface of AtATG12: one is conserved in both Atg12 and Atg8 orthologs, while the other is unique to Atg12 orthologs. Considering that they share Atg7 as an E1-like enzyme, we suggest that the first hydrophobic patch is responsible for the conjugation reaction, while the latter is involved in Atg12-specific functions.

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Section: Resultsmentioning

confidence: 99%

The Crystal Structure of Plant ATG12 and its Biological Implication in Autophagy

Suzuki

Yoshimoto²,

Fujioka³

et al. 2005

Autophagy

111

View full text Add to dashboard Cite

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“…Although a high resolution crystallographic structure for the heterodimer has been determined (26), a crystallographic structure for the Ub-bound tetramer is unlikely. This conclusion is based both on the instability of the hUbc13-Ub thiolester bond (36) and the relatively weak interaction that exists between the acceptor Ub and hMms2 (K d ϳ 100 M).…”

Section: Resultsmentioning

confidence: 99%

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

McKenna

Moraes

Pastushok

et al. 2003

Journal of Biological Chemistry

121

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A heterodimer composed of the catalytically active ubiquitin-conjugating enzyme hUbc13 and its catalytically inactive paralogue, hMms2, forms the catalytic core for the synthesis of an alternative type of multiubiquitin chain where ubiquitin molecules are tandemly linked to one another through a Lys-63 isopeptide bond. This type of linkage, as opposed to the more typical Lys-48-linked chains, serves as a non-proteolytic marker of protein targets involved in error-free postreplicative DNA repair and NF-B signal transduction. Using a two-dimensional 1 H-15 N NMR approach, we have mapped: 1) the interaction between the subunits of the human Ubc13⅐Mms2 heterodimer and 2) the interactions between each of the subunits or heterodimer with a non-covalently bound acceptor ubiquitin or a thiolesterlinked donor ubiquitin. Using these NMR-derived constraints and an unbiased docking approach, we have assembled the four components of this catalytic complex into a three-dimensional model that agrees well with its catalytic function.

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“…This is a remarkable improvement from other attempts to stabilize the ubiquitin-E2 linkage. For example, mutation of the active site cysteine in the E2, HsUbc2b, to a serine residue has allowed formation of an ester linkage between ubiquitin and the E2 (31)(32)(33). Although the resulting complex could be purified, it was short-lived at pH 6.7 and unstable at alkaline pH impeding any attempts to model the association of the two proteins or to use the complex for biochemical characterization (31 , and Leu 73 ) are marked with a square (see "Results" for details).…”

Section: Discussionmentioning

confidence: 99%

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

Merkley

Barber

Shaw

2005

Journal of Biological Chemistry

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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...

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Characterization of the binding interface between ubiquitin and class I human ubiquitin-conjugating enzyme 2b by multidimensional heteronuclear NMR spectroscopy in solution 1 1Edited by P. E. Wright

Cited by 92 publications

References 48 publications

The Crystal Structure of Plant ATG12 and its Biological Implication in Autophagy

The Crystal Structure of Plant ATG12 and its Biological Implication in Autophagy

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

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

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