Structure of HHARI, a RING-IBR-RING Ubiquitin Ligase: Autoinhibition of an Ariadne-Family E3 and Insights into Ligation Mechanism

Ubiquitination, the crucial posttranslational modification that regulates the eukaryotic proteome, is carried out by a trio of enzymes, known as E1 [ubiquitin (Ub)-activating enzyme], E2 (Ub-conjugating enzyme), and E3 (Ub ligase). Although most E2s can work with any of the three mechanistically distinct classes of E3s, the E2 UBCH7 is unable to function with really interesting new gene (RING)-type E3s, thereby restricting it to homologous to E6AP C-terminus (HECT) and RING-in-between-RING (RBR) E3s. The Caenorhabditis elegans UBCH7 homolog, UBC-18, plays a critical role in developmental processes through its cooperation with the RBR E3 ARI-1 (HHARI in humans). We discovered that another E2, ubc-3, interacts genetically with ubc-18 in an unbiased genome-wide RNAi screen in C. elegans. These two E2s have nonoverlapping biochemical activities, and each is dedicated to distinct classes of E3s. UBC-3 is the ortholog of CDC34 that functions specifically with Cullin-RING E3 ligases, such as SCF (Skp1-Cullin-F-box). Our genetic and biochemical studies show that UBCH7 (UBC-18) and the RBR E3 HHARI (ARI-1) coordinate with CDC34 (UBC-3) and an SCF E3 complex to ubiquitinate a common substrate, a SKP1-related protein. We show that UBCH7/HHARI primes the substrate with a single Ub in the presence of CUL-1, and that CDC34 is required to build chains onto the Ub-primed substrate. Our study reveals that the association and coordination of two distinct E2/E3 pairs play essential roles in a developmental pathway and suggests that cooperative action among E3s is a conserved feature from worms to humans.

Section: Genetic Interactions Between Ubc-18 and Genes Of The Scf E3supporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

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Two functionally distinct E2/E3 pairs coordinate sequential ubiquitination of a common substrate in Caenorhabditis elegans development

Dove

Kemp

Bona

et al. 2017

“…E3 ligases are regulated through diverse mechanisms; for example, the multi-subunit Cullin RING E3 ligases are regulated by posttranslational modification with NEDD8 (42), the HECT-class E3 ligase Smurf2 is regulated by intramolecular interactions between the HECT domain and its flanking N-terminal C2 domain (33), and the RBR-class E3 ligase HHARI is regulated by intramolecular interactions between the RBR module and its flanking C-terminal Ariadne domain (43). Not surprisingly, the IpaH family of E3 ligases, many of which are also regulated, have a completely different mechanism of autoregulation.…”

Section: Discussionmentioning

confidence: 99%

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

Keszei

Sicheri

2017

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 ...

“…These enzymes are structurally autoinhibited in their native states by unique accessory domains (11)(12)(13), indicating that RBR ligases must be activated to carry out their full ubiquitination potential. Specifically, parkin contains an N-terminal ubiquitin-like (UBL) domain shown to inhibit Ub ligase activity (11).…”

mentioning

confidence: 99%

Structure of phosphorylated UBL domain and insights into PINK1-orchestrated parkin activation

Aguirre

Dunkerley

Mercier

et al. 2016

Mutations in PARK2 and PARK6 genes are responsible for the majority of hereditary Parkinson's disease cases. These genes encode the E3 ubiquitin ligase parkin and the protein kinase PTENinduced kinase 1 (PINK1), respectively. Together, parkin and PINK1 regulate the mitophagy pathway, which recycles damaged mitochondria following oxidative stress. Native parkin is inactive and exists in an autoinhibited state mediated by its ubiquitin-like (UBL) domain. PINK1 phosphorylation of serine 65 in parkin's UBL and serine 65 of ubiquitin fully activate ubiquitin ligase activity; however, a structural rationale for these observations is not clear. Here, we report the structure of the phosphorylated UBL domain from parkin. We find that destabilization of the UBL results from rearrangements to hydrophobic core packing that modify its structure. Altered surface electrostatics from the phosphoserine group disrupt its intramolecular association, resulting in poorer autoinhibition in phosphorylated parkin. Further, we show that phosphorylation of both the UBL domain and ubiquitin are required to activate parkin by releasing the UBL domain, forming an extended structure needed to facilitate E2-ubiquitin binding. Together, the results underscore the importance of parkin activation by the PINK1 phosphorylation signal and provide a structural picture of the unraveling of parkin's ubiquitin ligase potential. . Multiple studies show that in response to mitochondrial oxidative stress parkin activity is stimulated through phosphorylation by PINK1 (4, 5). This in turn facilitates parkin-mediated ubiquitination of several proteins at the outer mitochondrial membrane and signals the turnover of damaged mitochondria through the mitophagy pathway (6-8). Some mutations in parkin cause its dysfunction, leading to an accumulation of mitochondrial damage that appears to be especially detrimental in neurons, a potential cause for Parkinson's disease.Parkin belongs to the RBR subfamily of E3 ubiquitin ligases (9) that function by transferring Ub from an E2-conjugating enzyme to a substrate through an E3-Ub intermediate (10). These enzymes are structurally autoinhibited in their native states by unique accessory domains (11-13), indicating that RBR ligases must be activated to carry out their full ubiquitination potential. Specifically, parkin contains an N-terminal ubiquitin-like (UBL) domain shown to inhibit Ub ligase activity (11). Threedimensional structures of parkin show UBL associates with the C-terminal region (R0RBR) through both ionic and hydrophobic interactions, blocking the proposed E2 recognition site (14, 15). PINK1 stimulates parkin activity through phosphorylation of both Ub and parkin's UBL domain at an equivalent serine 65 position in sequence and structure (16)(17)(18)(19)(20)(21). Whereas each phosphorylation event can increase parkin activity independently, maximal activity is obtained when both parkin and Ub are phosphorylated (18,19). Phosphorylation of parkin at S65 increases parkin's affinity for phosphoubiquitin (pUb) (1...