BIRC7–E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimer

Dou, Haoran; Buetow, Lori; Sibbet, Gary; Cameron, Kenneth; Huang, Danny T.

doi:10.1038/nsmb.2379

Cited by 286 publications

(474 citation statements)

References 56 publications

(104 reference statements)

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“…The structures of two dimeric RING E3s in complex with UBE2D‐Ub have been reported recently, and both show that the proximal RING binds the ubiquitin intermediate via ubiquitin's I36 surface patch. An aromatic residue from the distal RING makes additional contacts with the same ubiquitin surface, thereby stabilizing a “closed” conformation in which the E2~Ub is activated for transfer (Dou et al , 2012; Plechanovova et al , 2012). Similarly, in the TRIM25/E2~Ub structure each ubiquitin molecule is folded back onto its E2 in a conformation that resembles the one seen in other dimeric RING/E2~Ub complexes.…”

Section: Resultsmentioning

confidence: 99%

“…Similarly, R54 of TRIM25, referred to as the linchpin for allosteric activation of E2~Ub (Pruneda et al , 2012; Metzger et al , 2014), is involved in electrostatic interactions with Q92 of UBE2D1 and Q40 and R72 of ubiquitin, and accordingly, its mutation reduces catalytic activity in discharge assays and formation of K63‐linked chains by UBE2N/UBE2V1 (Fig 4B–D). In contrast, the aromatic residue from the distal RING (Y193 of RNF4 and F296 in BIRC7), that is important in other RINGs to engage ubiquitin in the closed conformation, does not exist in TRIM25 (Dou et al , 2012; Plechanovova et al , 2012). Instead, residues K65 and T67 of the RING domain contact D32 and E34 of the opposite, distal ubiquitin in one monomer, whereas in the other monomer, the K65 and D32 interaction is absent and replaced by a hydrogen bond between N71 of the RING and K33 of ubiquitin (Fig 4A).…”

Section: Resultsmentioning

confidence: 99%

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Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

et al. 2016

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TRIM E3 ubiquitin ligases regulate a wide variety of cellular processes and are particularly important during innate immune signalling events. They are characterized by a conserved tripartite motif in their N‐terminal portion which comprises a canonical RING domain, one or two B‐box domains and a coiled‐coil region that mediates ligase dimerization. Self‐association via the coiled‐coil has been suggested to be crucial for catalytic activity of TRIMs; however, the precise molecular mechanism underlying this observation remains elusive. Here, we provide a detailed characterization of the TRIM ligases TRIM25 and TRIM32 and show how their oligomeric state is linked to catalytic activity. The crystal structure of a complex between the TRIM25 RING domain and an ubiquitin‐loaded E2 identifies the structural and mechanistic features that promote a closed E2~Ub conformation to activate the thioester for ubiquitin transfer allowing us to propose a model for the regulation of activity in the full‐length protein. Our data reveal an unexpected diversity in the self‐association mechanism of TRIMs that might be crucial for their biological function.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

et al. 2016

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“…1A)-have been more closely examined for their role in ubiquitination mediated by distinct E2 ubiquitin-conjugating enzymes and E3 enzymes (e.g., refs. [22][23][24][25][26][27][28][29][30][31]. Notably, patch II but not patch I was shown to be required for covalent attachment of ubiquitin to substrate proteins catalyzed by the HECT E3s Rsp5 and NEDD4L (22,30).…”

Section: Resultsmentioning

confidence: 99%

Role of ubiquitin and the HPV E6 oncoprotein in E6AP-mediated ubiquitination

Mortensen

Schneider

Barbic

et al. 2015

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

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Deregulation of the ubiquitin ligase E6 associated protein (E6AP) encoded by the UBE3A gene has been associated with three different clinical pictures. Hijacking of E6AP by the E6 oncoprotein of distinct human papillomaviruses (HPV) contributes to the development of cervical cancer, whereas loss of E6AP expression or function is the cause of Angelman syndrome, a neurodevelopmental disorder, and increased expression of E6AP has been involved in autism spectrum disorders. Although these observations indicate that the activity of E6AP has to be tightly controlled, only little is known about how E6AP is regulated at the posttranslational level. Here, we provide evidence that the hydrophobic patch of ubiquitin comprising Leu-8 and Ile-44 is important for E6AP-mediated ubiquitination, whereas it does not affect the catalytic properties of the isolated catalytic HECT domain of E6AP. Furthermore, we show that the HPV E6 oncoprotein rescues the disability of full-length E6AP to use a respective hydrophobic patch mutant of ubiquitin for ubiquitination and that it stimulates E6AP-mediated ubiquitination of Ring1B, a known substrate of E6AP, in vitro and in cells. Based on these data, we propose that E6AP exists in at least two different states, an active and a less active or latent one, and that the activity of E6AP is controlled by noncovalent interactions with ubiquitin and allosteric activators such as the HPV E6 oncoprotein.I n eukaryotes, posttranslational modification of proteins by ubiquitin plays a pivotal role in the regulation of many cellular processes, including cell cycle, DNA metabolism (e.g., DNA repair, transcription), and various signal transduction pathways (1-4). The specificity of the ubiquitin-conjugation system is mainly ensured by E3 ubiquitin ligases, which mediate the recognition of target proteins. Based on the presence of distinct domains and their mode of action, E3 proteins can be grouped into three families, RING/RING-like E3s, RING-in-between-RING (RBR) E3s, and HECT E3s (5-7). All E3s have interaction sites for both substrate proteins and E2 ubiquitinconjugating enzymes. However, whereas in the case of RBR E3s and HECT E3s, ubiquitin is transferred from the E3 to substrates, RING/RING-like E3s function as adaptors between substrates and E2s (i.e., ubiquitin is transferred from the E2 to the substrate).E6AP, the founding member of the HECT E3 family, was originally identified as an interacting protein of the E6 oncoprotein of cancer-associated human papillomaviruses (HPVs) (8, 9). The E6-E6AP complex targets the tumor suppressor p53 and other proteins-which in the absence of E6 are not targeted by E6AP-for ubiquitination and degradation thereby contributing to HPV-induced cervical carcinogenesis (10, 11). In 1997, it was recognized that alterations in the UBE3A gene, which encodes E6AP, resulting in loss of E6AP expression or in the expression of E6AP variants with compromised E3 activity, are the cause of the Angelman syndrome (AS), a neurodevelopmental disorder (12-14). Recently, it was rep...

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“…In several instances, it has been shown that dimeric E3s require dimerization for ubiquitination activity (Dueber et al, 2011;Dou et al, 2012a;Plechanovová et al, 2012). Elegant experiments by Plechanovová et al (2012) and Dou et al (2012b) showed that the E2 contacts a single protomer of the dimeric E3 (RNF4 and Birc7, respectively), while ubiquitin is folded back onto the E2 via contacts from both RING molecules.…”

Section: Dimerization and E3 Ligase Activitymentioning

confidence: 99%

“…In several instances, homooligomerization was shown to influence the activity of both self and substrate ubiquitination. Mutant variants that were impaired in their oligomerization displayed reduced activity (Yin et al, 2009;Dueber et al, 2011;Plechanovová et al, 2011;Dou et al, 2012a). Exceptions include the U-box E3s E4B in humans and its ortholog Ufd2 in yeast (Saccharomyces cerevisiae), which function as monomers (Benirschke et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Changes in PUB22 Ubiquitination Modes Triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 Dampen the Immune Response

et al. 2017

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Crosstalk between posttranslational modifications, such as ubiquitination and phosphorylation, play key roles in controlling the duration and intensity of signaling events to ensure cellular homeostasis. However, the molecular mechanisms underlying the regulation of negative feedback loops remain poorly understood. Here, we uncover a pathway in Arabidopsis thaliana by which a negative feedback loop involving the E3 ubiquitin ligase PUB22 that dampens the immune response is triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3), best known for its function in the activation of signaling. PUB22's stability is controlled by MPK3-mediated phosphorylation of residues localized in and adjacent to the E2 docking domain. We show that phosphorylation is critical for stabilization by inhibiting PUB22 oligomerization and, thus, autoubiquitination. The activity switch allows PUB22 to dampen the immune response. This regulatory mechanism also suggests that autoubiquitination, which is inherent to most single unit E3s in vitro, can function as a self-regulatory mechanism in vivo.

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BIRC7–E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimer

Cited by 286 publications

References 56 publications

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

Role of ubiquitin and the HPV E6 oncoprotein in E6AP-mediated ubiquitination

Changes in PUB22 Ubiquitination Modes Triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 Dampen the Immune Response

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