Structural basis for the RING catalyzed synthesis of K63 linked ubiquitin chains

This work was supported by grants from Cancer Research UK (C434/A13067), the Wellcome Trust (098391/Z/12/Z) and Biotechnology and Biological Sciences Research Council (BB/J016004/1).The RING E3 ligase catalysed formation of lysine 63 linked ubiquitin chains by the Ube2V2–Ubc13 E2 complex is required for many important biological processes. Here we report the structure of the RING domain dimer of rat RNF4 in complex with a human Ubc13~Ub conjugate and Ube2V2. The structure has captured Ube2V2 bound to the acceptor (priming) ubiquitin with Lys63 in a position that could lead to attack on the linkage between the donor (second) ubiquitin and Ubc13 that is held in the active “folded back” conformation by the RING domain of RNF4. The interfaces identified in the structure were verified by in vitro ubiquitination assays of site directed mutants. This represents the first view of the synthesis of Lys63 linked ubiquitin chains in which both substrate ubiquitin and ubiquitin-loaded E2 are juxtaposed to allow E3 ligase mediated catalysis.PostprintPeer reviewe

Structural analysis of MDM2 RING separates degradation from regulation of p53 transcription activity

MDM2–MDMX complexes bind the p53 tumor-suppressor protein, inhibiting p53's transcriptional activity and targeting p53 for proteasomal degradation. Inhibitors that disrupt binding between p53 and MDM2 efficiently activate a p53 response, but their use in the treatment of cancers that retain wild-type p53 may be limited by on-target toxicities due to p53 activation in normal tissue. Guided by a novel crystal structure of the MDM2–MDMX–E2(UbcH5B)–ubiquitin complex, we designed MDM2 mutants that prevent E2–ubiquitin binding without altering the RING-domain structure. These mutants lack MDM2's E3 activity but retain the ability to limit p53′s transcriptional activity and allow cell proliferation. Cells expressing these mutants respond more quickly to cellular stress than cells expressing wild-type MDM2, but basal p53 control is maintained. Targeting the MDM2 E3-ligase activity could therefore widen the therapeutic window of p53 activation in tumors

Human BRCA1-BARD1 ubiquitin ligase activity counters chromatin barriers to DNA resection

The opposing activities of 53BP1 and BRCA1 influence pathway choice of DNA double-strand break repair. How BRCA1 counters the inhibitory effect of 53BP1 on DNA resection and homologous recombination is unknown. Here we identify the site of BRCA1-BARD1 required for priming ubiquitin transfer from E2~ubiquitin. We demonstrate that BRCA1-BARD1’s ubiquitin ligase activity is required for repositioning 53BP1 on damaged chromatin. We confirm H2A ubiquitylation by BRCA1-BARD1 and show that an H2A-ubiquitin fusion protein promotes DNA resection and repair in BARD1 deficient cells. We show BRCA1-BARD1 function in homologous recombination requires the chromatin remodeler SMARCAD1. SMARCAD1 binding to H2A-ubiquitin, optimal localization to sites of damage and activity in DNA repair requires its ubiquitin-binding CUE domains. SMARCAD1 is required for 53BP1 repositioning and the need for SMARCAD1 in Olaparib or camptothecin resistance is alleviated by 53BP1 loss. Thus BRCA1- BARD1 ligase activity and subsequent SMARCAD1-dependent chromatin remodeling are critical regulators of DNA repair

Activity-based E3 ligase profiling uncovers an E3 ligase with esterification activity

Ubiquitination is initiated by transfer of ubiquitin (Ub) from a ubiquitin-activating enzyme (E1) to a ubiquitin-conjugating enzyme (E2), producing a covalently linked intermediate (E2-Ub)(1). Ubiquitin ligases (E3s) of the 'really interesting new gene' (RING) class recruit E2-Ub via their RING domain and then mediate direct transfer of ubiquitin to substrates(2). By contrast, 'homologous to E6-AP carboxy terminus' (HECT) E3 ligases undergo a catalytic cysteine-dependent transthiolation reaction with E2-Ub, forming a covalent E3-Ub intermediate(3,4). Additionally, RING-between-RING (RBR) E3 ligases have a canonical RING domain that is linked to an ancillary domain. This ancillary domain contains a catalytic cysteine that enables a hybrid RING-HECT mechanism(5). Ubiquitination is typically considered a post-translational modification of lysine residues, as there are no known human E3 ligases with non-lysine activity. Here we perform activity-based protein profiling of HECT or RBR-like E3 ligases and identify the neuron-associated E3 ligase MYCBP2 (also known as PHR1) as the apparent single member of a class of RING-linked E3 ligase with esterification activity and intrinsic selectivity for threonine over serine. MYCBP2 contains two essential catalytic cysteine residues that relay ubiquitin to its substrate via thioester intermediates. Crystallographic characterization of this class of E3 ligase, which we designate RING-Cys-relay (RCR), provides insights into its mechanism and threonine selectivity. These findings implicate non-lysine ubiquitination in cellular regulation of higher eukaryotes and suggest that E3 enzymes have an unappreciated mechanistic diversity

Ubiquitin transfer by a RING E3 ligase occurs from a closed E2~ubiquitin conformation

Funding: Investigator Award from the Wellcome Trust (098391/Z/12/Z) and (217196/Z/19/Z) and a Programme grant from Cancer Research UK (C434/A21747) to R.T.H.; J.C.P. thanks the University of St Andrews for financial support.Based on extensive structural analysis it was proposed that RING E3 ligases prime the E2~ubiquitin conjugate (E2~Ub) for catalysis by locking it into a closed conformation, where ubiquitin is folded back onto the E2 exposing the restrained thioester bond to attack by substrate nucleophile. However the proposal that the RING dependent closed conformation of E2~Ub represents the active form that mediates ubiquitin transfer has yet to be experimentally tested. To test this hypothesis we use single molecule Förster Resonance Energy Transfer (smFRET) to measure the conformation of a FRET labelled E2~Ub conjugate, which distinguishes between closed and alternative conformations. We describe a real-time FRET assay with a thioester linked E2~Ub conjugate to monitor single ubiquitination events and demonstrate that ubiquitin is transferred to substrate from the closed conformation. These findings are likely to be relevant to all RING E3 catalysed reactions ligating ubiquitin and other ubiquitin-like proteins (Ubls) to substrates.Publisher PDFPeer reviewe

Structure of a ubiquitin-loaded HECT ligase reveals the molecular basis for catalytic priming

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RBR ligase–mediated ubiquitin transfer: a tale with many twists and turns

RBR ligases are an enigmatic class of E3 ubiquitin ligases that combine properties of RING and HECT-type E3s and undergo multilevel regulation through autoinhibition, post-translational modifications, multimerization and interaction with binding partners. Here, we summarize recent progress in RBR structures and function, which has uncovered commonalities in the mechanisms by which different family members transfer ubiquitin through a multistep process. However, these studies have also highlighted clear differences in the activity of different family members, suggesting that each RBR ligase has evolved specific properties to fit the biological process it regulates

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

RING E3 ligases catalyze the transfer of ubiquitin (Ub) from E2 ubiquitin-conjugating enzyme thioesterified with Ub (E2~Ub) to substrate. For RING E3 dimers, the RING domain of one subunit and tail of the second cooperate to prime Ub, but how this is accomplished by monomeric RING E3s in the absence of a tail-like component is currently unknown. Here, we present a crystal structure of a monomeric RING E3, Tyr363-phosphorylated human CBL-B, bound to a stabilized Ub-linked E2, revealing a similar mechanism in activating E2~Ub. Both pTyr363 and the pTyr363-induced element interact directly with Ub's Ile36 surface, improving the catalytic efficiency of Ub transfer by ~200-fold. Hence, interactions outside the canonical RING domain are crucial for optimizing Ub transfer in both monomeric and dimeric RING E3s. We propose that an additional non-RING Ub-priming element may be a common RING E3 feature