Structural and Functional Characterization of the Monomeric U-Box Domain from E4B

Nordquist, Kyle A.; Dimitrova, Yoana N.; Brzović, Peter S.; Ridenour, Whitney B.; Munro, Kim; Soss, Sarah E.; Caprioli, Richard M.; Klevit, Rachel E.; Chazin, Walter J.

doi:10.1021/bi901620v

Cited by 34 publications

(37 citation statements)

References 38 publications

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“…6A). Addition of 0.25 mol equivalents of each E4BU variant (L1107I, M1124V, D1139N, and N1142T) to 15 N-UbcH5c-O∼Ub increased the magnitude of CSPs in these resonances beyond those observed for addition of WT E4BU (Fig. 6 D and E).…”

Section: Mechanistic Classification Of Mutations That Enhance E3 Ligasementioning

confidence: 86%

“…S5C). The capacity of each E4BU variant to bind UbcH5c was assessed using 1 H, 15 N HSQC transverse relaxation-optimized spectroscopy (TROSY) experiments in which increasing quantities of E4BU were titrated into 15 N-UbcH5c. The spectra confirm that each E4BU variant binds to the same E2 surface composed of residues in helix 1, loop 4, and loop 7 of UbcH5c (Fig.…”

Section: Mechanistic Classification Of Mutations That Enhance E3 Ligasementioning

confidence: 99%

“…We adapted this method to generate a sequence-function map of the U-box of the murine E3 ligase ubiquitination factor E4B (Ube4b). The U-box domain of Ube4b is an ideal candidate for this approach because it functions as a monomer; its structure has been solved both in isolation and in complex with the E2 UbcH5c; and with an extended N-terminal region, it exhibits auto-ubiquitination activity in vitro (14,15). In vivo, the human homolog UBE4B polyubiquitinates the tumor suppressor p53 to target it for degradation by the proteasome.…”

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confidence: 99%

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Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis

Starita

Pruneda

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Although ubiquitination plays a critical role in virtually all cellular processes, mechanistic details of ubiquitin (Ub) transfer are still being defined. To identify the molecular determinants within E3 ligases that modulate activity, we scored each member of a library of nearly 100,000 protein variants of the murine ubiquitination factor E4B (Ube4b) U-box domain for auto-ubiquitination activity in the presence of the E2 UbcH5c. This assay identified mutations that enhance activity both in vitro and in cellular p53 degradation assays. The activityenhancing mutations fall into two distinct mechanistic classes: One increases the U-box:E2-binding affinity, and the other allosterically stimulates the formation of catalytically active conformations of the E2∼Ub conjugate. The same mutations enhance E3 activity in the presence of another E2, Ube2w, implying a common allosteric mechanism, and therefore the general applicability of our observations to other E3s. A comparison of the E3 activity with the two different E2s identified an additional variant that exhibits E3:E2 specificity. Our results highlight the general utility of high-throughput mutagenesis in delineating the molecular basis of enzyme activity.NMR | phage display | ubiquitin E3 ligase | protein stability | deep mutational scanning C ovalent modification of proteins by ubiquitin (Ub) has an impact on nearly all eukaryotic cell biology. The attachment of Ub is accomplished by three enzymes: an E1 Ub-activating enzyme, an E2 Ub-conjugating enzyme, and an E3 Ub ligase (1). In the final stage of the pathway, the E3 ligase facilitates transfer from a Ub-loaded E2 (termed an E2∼Ub conjugate) onto a substrate lysine. Minimally, functional E3s contain an E2-binding domain and a substrate-recognition domain, enabling them to bind an E2∼Ub and a substrate simultaneously. The majority of E3s harbor either a RING (really interesting new gene) domain or a related U-box domain to bind cognate E2s. RING-type E3s enhance transfer of Ub directly from an E2's active site to a substrate lysine without an intermediate transfer step of Ub to the E3 itself, as occurs with homology to E6AP carboxyl-terminus (HECT)-type ligases. In addition to providing proximity between the Ub attached to the active site of an E2 and a substrate amino group, RING-type E3s activate E2∼Ub conjugates allosterically (2-11). Allosteric activation relies on promotion of catalytically active "closed" conformations of an E2∼Ub conjugate that presumably arrange the E2 active site thioester for access and attack by an incoming nucleophile. Thus, two processes contribute to the rate enhancement of Ub transfer by RING-type E3s: (i) proximity (and, in some cases, orientational) effects and (ii) promotion of reactive states of the E2∼Ub. Recent studies (8-10) have shown that minimal RING-type domains that lack a substrate-binding activity (and therefore cannot provide a proximity enhancement) are able to enhance the intrinsic reactivity of E2∼Ub species, demonstrating that the two sources of rate enhancemen...

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Section: Mechanistic Classification Of Mutations That Enhance E3 Ligasementioning

confidence: 86%

Section: Mechanistic Classification Of Mutations That Enhance E3 Ligasementioning

confidence: 99%

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confidence: 99%

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Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis

Starita

Pruneda

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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174

145

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“…Ufd2 is a prototypical E4 ubiquitin chain elongating enzyme and Cdc48 substrate-processing cofactor (35,56). Nevertheless, its ability to directly interact with E2 ubiquitin-conjugating enzymes (46) as well as with the ubiquitin binding protein Rad23 (24,55) raised the possibility that Ufd2 may bind and multiubiquitylate preubiquitylated substrates independently of Cdc48. However, the phenotypes of the cdc48 mutants clearly indicate that Ufd2 cannot function independently of Cdc48 in the OLE and UFD protein degradation pathways ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Cellular Functions of Ufd2 and Ufd3 in Proteasomal Protein Degradation Depend on Cdc48 Binding

Böhm

Lamberti

Fernández‐Sáiz

et al. 2011

Molecular and Cellular Biology

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The chaperone-related AAA ATPase Cdc48 (p97/VCP in higher eukaryotes) segregates ubiquitylated proteins for subsequent degradation by the 26S proteasome or for nonproteolytic fates. The specific outcome of Cdc48 activity is controlled by the evolutionary conserved cofactors Ufd2 and Ufd3, which antagonistically regulate the substrates' ubiquitylation states. In contrast to the interaction of Ufd3 and Cdc48, the interaction between the ubiquitin chain elongating enzyme Ufd2 and Cdc48 has not been precisely mapped. Consequently, it is still unknown whether physiological functions of Ufd2 in fact require Cdc48 binding. Here, we show that Ufd2 binds to the C-terminal tail of Cdc48, unlike the human Ufd2 homologue E4B, which interacts with the N domain of p97. The binding sites for Ufd2 and Ufd3 on Cdc48 overlap and depend critically on the conserved residue Y834 but are not identical. Saccharomyces cerevisiae cdc48 mutants altered in residue Y834 or lacking the C-terminal tail are viable and exhibit normal growth. Importantly, however, loss of Ufd2 and Ufd3 binding in these mutants phenocopies defects of ⌬ufd2 and ⌬ufd3 mutants in the ubiquitin fusion degradation (UFD) and Ole1 fatty acid desaturase activation (OLE) pathways. These results indicate that key cellular functions of Ufd2 and Ufd3 in proteasomal protein degradation require their interaction with Cdc48.

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“…U-box E3 ligases function as scaffolds for ubiquitination by recruiting E2 conjugating enzymes to the U-box domain and binding target substrate proteins through other domains present in the protein (Zhang et al, 2005;Xu et al, 2008;Nordquist et al, 2010). Exo70A1 was identified as a target of ARC1 and is ubiquitinated by ARC1 (Samuel et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

The ARC1 E3 Ligase Promotes Two Different Self-Pollen Avoidance Traits inArabidopsis

2014

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Flowering plants have evolved various strategies for avoiding self-pollen to drive genetic diversity. These strategies include spatially separated sexual organs (herkogamy), timing differences between male pollen release and female pistil receptivity (dichogamy), and self-pollen rejection. Within the Brassicaceae, these outcrossing systems are the evolutionary default state, and many species display these traits, including Arabidopsis lyrata. In contrast to A. lyrata, closely related Arabidopsis thaliana has lost these self-pollen traits and thus represents an excellent system to test genes for reconstructing these evolutionary traits. We previously demonstrated that the ARC1 E3 ligase is required for self-incompatibility in two diverse Brassicaceae species, Brassica napus and A. lyrata, and is frequently deleted in self-compatible species, including A. thaliana. In this study, we examined ARC1's requirement for reconstituting self-incompatibility in A. thaliana and uncovered an important role for ARC1 in promoting a strong and stable pollen rejection response when expressed with two other A. lyrata self-incompatibility factors. Furthermore, we discovered that ARC1 promoted an approach herkogamous phenotype in A. thaliana flowers. Thus, ARC1's expression resulted in two different A. lyrata traits for self-pollen avoidance and highlights the key role that ARC1 plays in the evolution and retention of outcrossing systems.

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Structural and Functional Characterization of the Monomeric U-Box Domain from E4B

Cited by 34 publications

References 38 publications

Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis

Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis

Cellular Functions of Ufd2 and Ufd3 in Proteasomal Protein Degradation Depend on Cdc48 Binding

The ARC1 E3 Ligase Promotes Two Different Self-Pollen Avoidance Traits inArabidopsis

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