Structural basis for ligase-specific conjugation of linear ubiquitin chains by HOIP

Stieglitz, Benjamin; Rana, Rohini R.; Koliopoulos, Marios G.; Morris-Davies, Aylin C.; Schaeffer, Véronique; Christodoulou, Evangelos; Howell, Steven; Brown, Nicholas R.; Đikić, Ivan; Rittinger, Katrin

doi:10.1038/nature12638

Cited by 185 publications

(265 citation statements)

References 37 publications

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“…The Ub conjugate is positioned such that two hydrophobic regions including the I44 patch and the C‐terminus (V70, L71) of Ub are pointed away from the RING1 domain and exposed to solvent. Although not identical, the activated Ub in the HOIP/UbcH5b complex and donor Ub in the RING2L transfer complex are positioned similarly (Stieglitz et al , 2013; Lechtenberg et al , 2016). These hydrophobic regions (I44, V70, L71) are used to recruit helix h L2 from the catalytic RING2L domain in the domain‐swapped dimer structure (Lechtenberg et al , 2016).…”

Section: Resultsmentioning

confidence: 99%

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation

et al. 2018

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The E3 ligase parkin ubiquitinates outer mitochondrial membrane proteins during oxidative stress and is linked to early‐onset Parkinson's disease. Parkin is autoinhibited but is activated by the kinase PINK1 that phosphorylates ubiquitin leading to parkin recruitment, and stimulates phosphorylation of parkin's N‐terminal ubiquitin‐like (pUbl) domain. How these events alter the structure of parkin to allow recruitment of an E2~Ub conjugate and enhanced ubiquitination is an unresolved question. We present a model of an E2~Ub conjugate bound to the phospho‐ubiquitin‐loaded C‐terminus of parkin, derived from NMR chemical shift perturbation experiments. We show the UbcH7~Ub conjugate binds in the open state whereby conjugated ubiquitin binds to the RING1/IBR interface. Further, NMR and mass spectrometry experiments indicate the RING0/RING2 interface is re‐modelled, remote from the E2 binding site, and this alters the reactivity of the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer. Our experiments provide evidence that parkin phosphorylation and E2~Ub recruitment act synergistically to enhance a weak interaction of the pUbl domain with the RING0 domain and rearrange the location of the RING2(Rcat) domain to drive parkin activity.

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Section: Resultsmentioning

confidence: 99%

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation

et al. 2018

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“…The UBE2D~Ub intermediates were synthesized as previously described (Stieglitz et al , 2013). Briefly, His‐Ube1 (1 μM), UBE2D3 or UBE2D1 (250 μM), Ub or Atto 647N‐Ub (Ub Atto ) (500 μM) and ATP (3 mM) (Sigma) were incubated in reaction buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 20 mM MgCl 2 for 60 min at 25°C.…”

Section: Methodsmentioning

confidence: 99%

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 this context, an outwardly normal Ub discharge reaction in the absence of a deprotonated lysine acceptor can be achieved via nucleophilic attack by solvent molecules, yielding nonproductive hydrolysis. Importantly, the resultant defect in aminolysis can be partially rescued by the nonspecific deprotonation of an attacking lysine side chain through an increase in the solution pH (30). In contrast, mutation of the catalytic acid produces an exaggeratedly more stable E3∼Ub/E2∼Ub intermediate owing to impaired discharge function resulting from insufficient stabilization of the tetrahedral intermediate formed by an attacking nucleophile (27)(28)(29).…”

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

confidence: 99%

“…The participation of specific residues in either function can be discerned by assaying the effect of mutation on the penultimate steps of Ub thioester charging and discharging of the E2/E3 enzyme and on Ub-acceptor bond formation. For example, mutation of the catalytic base can allow apparently normal E3∼Ub charging and discharging, but with a failure to form Ub chains owing to a lack of nucleophilic activation of the substrate lysine (27)(28)(29)(30). In this context, an outwardly normal Ub discharge reaction in the absence of a deprotonated lysine acceptor can be achieved via nucleophilic attack by solvent molecules, yielding nonproductive hydrolysis.…”

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

confidence: 99%

“…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%

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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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Structural basis for ligase-specific conjugation of linear ubiquitin chains by HOIP

Cited by 185 publications

References 37 publications

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation

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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