Essentiality of a non-RING element in priming donor ubiquitin for catalysis by a monomeric E3

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

doi:10.1038/nsmb.2621

Cited by 121 publications

(163 citation statements)

References 45 publications

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“…Through structural and mutagenesis studies, we identify a novel and conserved mechanism through which canonical PCGF2/4 limits the activity of RING1. In addition, we demonstrate that, contrary to expectation 6,30 , PCGF-RING1 ligases use a mode of Ub recognition and activation 31 highly similar to that recently discovered for homodimeric RING E3s 30,32,33 and monomeric RINGs CBL-B and RNF38 34,35 . In contrast to the differences in their intrinsic catalytic activities, we find all the six PCGF-RING1 heterodimers to be robust H2A ligases in an assay using purified components.…”

supporting

confidence: 63%

BMI1–RING1B is an autoinhibited RING E3 ubiquitin ligase

Taherbhoy

Huang²,

Cochran³

2015

Nat Commun

106

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Polycomb repressive complex 1 (PRC1) is required for ubiquitination of histone H2A lysine 119, an epigenetic mark associated with repression of genes important in developmental regulation. The E3 ligase activity of PRC1 resides in the RING1A/B subunit when paired with one of six PCGF partners. The best known of these is the oncogene BMI1/PCGF4. We find that canonical PRC1 E3 ligases such as PCGF4-RING1B have intrinsically very low enzymatic activity compared with non-canonical PRC1 RING dimers. The structure of a high-activity variant in complex with E2 (PCGF5-RING1B-UbcH5c) reveals only subtle differences from an earlier PCGF4 complex structure. However, two charged residues present in the modelled interface with E2-conjugated ubiquitin prove critical: in BMI1/PCGF4, these residues form a salt bridge that may limit efficient ubiquitin transfer. The intrinsically low activity of the PCGF4-RING1B heterodimer is offset by a relatively favourable interaction with nucleosome substrates, resulting in an efficient site-specific monoubiquitination.

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supporting

confidence: 63%

BMI1–RING1B is an autoinhibited RING E3 ubiquitin ligase

Taherbhoy

Huang²,

Cochran³

2015

Nat Commun

106

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“…Moreover, structural and sequence comparison of the RINGs of TRIM25 and TRIM5α shows that E11 and E12 occupy similar positions (Yudina et al , 2015). Intriguingly, structural alignment of the RINGs of TRIM25 and monomeric Cbl‐b shows that Y363 of Cbl‐b, which needs to become phosphorylated for full ligase activity, is in the equivalent position of E10, although the residues come from different structural elements (Dou et al , 2013). This suggests that both monomeric and dimeric E3s may make use of an acidic element to stabilize the active E2~Ub conformation.…”

Section: Resultsmentioning

confidence: 99%

“…Importantly, the structure of the TRIM25 RING/E2~Ub complex shows how dimerization is used to enhance the rate of ubiquitin transfer: by stabilization of a closed E2~Ub conformation as observed previously for the dimeric RINGs RNF4 and BIRC7 (Dou et al , 2012; Plechanovova et al , 2012). In addition, our structure highlights that TRIM25 uses an additional feature to enforce the closed conformation that is reminiscent of the monomeric E3 Cbl‐b that uses a phospho‐tyrosine outside the RING domain to stabilize the E2~ubiquitin intermediate (Dou et al , 2013). There are no reports of phosphorylation events adjacent to the RING domain regulating TRIM catalytic activity, and instead, TRIM25 uses a glutamate in the N‐terminal helix to contact the opposite ubiquitin in a spatial arrangement that is highly similar to the pTyr of Cbl‐b.…”

Section: Discussionmentioning

confidence: 99%

“…Dimeric RINGs stabilize the closed E2~Ub conformation by using both RINGs in the dimer to contact the ubiquitin of the E2‐intermediate simultaneously (Dou et al , 2012; Plechanovova et al , 2012). Monomeric RINGs can also stabilize a closed conformation; however, they do so using different structural elements outside the core RING domain (Dou et al , 2013). …”

Section: Introductionmentioning

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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“…In general (and as best worked out for E2 enzymes), aminolysis between Ub charged onto an E2 or E3 enzyme and a lysine acceptor involves general acid-base catalysis (27)(28)(29)(30)(31). Within the active site, a catalytic base acts to deprotonate the incoming substrate lysine to promote its nucleophilic potential.…”

Section: D339 Functions As a Catalytic Acid And D397 Functions As A Cmentioning

confidence: 99%

Mechanism of catalysis, E2 recognition, and autoinhibition for the IpaH family of bacterial E3 ubiquitin ligases

Keszei

Sicheri

2017

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

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IpaH enzymes are secreted bacterial effectors that function within host cells as E3 ubiquitin (Ub) ligases. Catalytic activity is imparted by a conserved novel E3 ligase (NEL) domain that is unique to Gram-negative pathogens and whose activity is repressed by a flanking substrate-binding leucine-rich repeat (LRR) domain when substrate is absent. How the NEL domain catalyzes the conjugation of Ub onto substrates, recognizes host E2s, and maintains its autoinhibited state remain poorly understood. Here we used mutagenesis and enzyme kinetic analyses to address these gaps in knowledge. Mutagenesis of conserved residues on two remote surfaces of the NEL domain identified functional clusters proximal to and distal to the active site cysteine. By analyzing the kinetics of Ub charging and discharging, we identified proximal active site residues that function as either the catalytic acid or catalytic base for aminolysis. Further analysis revealed that distal site residues mediate the direct binding of E2. In studying the full-length protein, we also have uncovered that IpaH family autoinhibition is achieved by a short-circuiting mechanism wherein the LRR domain selectively blocks productive aminolysis, but not the nonproductive discharge of Ub from the E3 to solvent. This mode of autoinhibition, which is not shared by the HECT domain ligase Smurf2, leads to the unanticipated depletion of E2∼Ub and thus a concomitant dominant-negative effect on other E3s in vitro, raising the possibility that short circuiting also may serve to restrict the function of host E3s in cells.IpaH family | bacterial E3 ubiquitin ligase | ubiquitin | host-pathogen P osttranslational modification by the covalent attachment of the small protein ubiquitin (Ub) onto lysine side chains of target proteins is an essential eukaryotic modification that imparts a wide range of consequences to the substrate, from targeting to the proteasome for degradation to the assembly of signaling complexes (1, 2). Ubiquitination begins with an E1 enzyme, which activates Ub through the consumption of ATP to form a thioester E1∼Ub adduct on its catalytic cysteine. The E1∼Ub conjugate then transthiolates Ub to the active-site cysteine of an E2 enzyme to form an E2∼Ub adduct. Finally, the charged E2∼Ub then collaborates with a diverse group of E3 ligases to catalyze the stable attachment of Ub to the e-amino group of side-chain lysine residues or, in some cases, the N-terminal amino group of protein substrates via an aminolysis reaction.E3 ligases can be divided into two groups based on whether or not they use a catalytic cysteine to form an E3∼Ub intermediate. The HECT (homologous to E6AP C-terminus), RBR (RING-inbetween-RING), and IpaH (invasion-plasmid antigen H) families of structurally distinct E3 ligases use a catalytic cysteine residue to accept activated Ub in the form of an E3∼Ub thioester adduct. The E3 itself then catalyzes the aminolysis reaction to form an isopeptide bond between Ub and the acceptor lysine substrate. In contrast, the RING (really interesting ...

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Essentiality of a non-RING element in priming donor ubiquitin for catalysis by a monomeric E3

Cited by 121 publications

References 45 publications

BMI1–RING1B is an autoinhibited RING E3 ubiquitin ligase

BMI1–RING1B is an autoinhibited RING E3 ubiquitin ligase

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

Mechanism of catalysis, E2 recognition, and autoinhibition for the IpaH family of bacterial E3 ubiquitin ligases

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