Summary Ubiquitination is a post-translational modification that regulates a myriad of cellular processes in eukaryotes1–4. The conventional ubiquitination cascade culminates in a covalent linkage between the C-terminus of ubiquitin (Ub) and a target protein, most often on a lysine sidechain1,5. Recent studies of the Legionella pneumophila SidE family of effector proteins revealed a novel mode of ubiquitination in which a phosphoribosylated ubiquitin (PR-Ub) is conjugated to a serine residue on substrates via a phosphodiester bond6–8. To uncover the molecular mechanism of this unique post-translational modification, we determined the crystal structure of a fragment of the SidE family member SdeA that retains ubiquitination activity. The structure reveals that the catalytic module contains two distinct functional units: a phosphodiesterase domain (PDE) and a mono-ADP-ribosyltransferase (mART) domain. Biochemical analysis shows that the mART domain-mediated conversion of Ub to ADP-ribosylated Ub (ADPR-Ub) and the PDE domain-mediated ligation of PR-Ub to substrates are two independent activities of SdeA. Furthermore, we present two crystal structures of a homologous PDE domain from the SidE family member SdeD9 in complex with Ub or ADPR-Ub. The structures suggest an intriguing mechanism for how SdeA processes ADPR-Ub to PR-Ub plus AMP and conjugates PR-Ub to a serine residue in substrates. Our study establishes the molecular mechanism of phosphoribosyl-ubiquitination (PR-ubiquitination) and paves the way for future studies of this unusual type of ubiquitination in eukaryotes.

SummaryRING-between-RING (RBR) E3s contain RING1 domains that are structurally similar yet mechanistically distinct from canonical RING domains. Both types of E3 bind E2∼ubiquitin (E2∼Ub) via their RINGs but canonical RING E3s promote closed E2∼Ub conformations required for direct Ub transfer from the E2 to substrate, while RBR RING1s promote open E2∼Ub to favor Ub transfer to the E3 active site. This different RING/E2∼Ub conformation determines its direct target, which for canonical RING E3s is typically a substrate or substrate-linked Ub, but is the E3 active-site cysteine in the case of RBR-type E3s. Here we show that a short extension of HHARI RING1, namely Zn2+-loop II, not present in any RING E3s, acts as a steric wedge to disrupt closed E2∼Ub, providing a structural explanation for the distinctive RING1-dependent conformational restriction mechanism utilized by RBR E3s.

Background:The sumoylation pathway is conserved in Plasmodium falciparum. Results: The small ubiquitin-related modifier (SUMO) E1 and E2 enzymes are not functionally interchangable between humans and the malaria parasite, P. falciparum. Conclusion: P. falciparum E1 and E2 interactions have significantly diverged from humans. Significance: Divergent E1 and E2 interaction could be exploited for the design of parasite specific inhibitors.

Protein modification by small ubiquitin-related modifiers (SUMOs) is essential and conserved in the malaria parasite, Plasmodium falciparum. We have previously shown that interactions between the SUMO E1-activating and E2-conjugating enzyme in P. falciparum are distinct compared with human, suggesting a potential target for development of parasite-specific inhibitors of SUMOylation. The parasite asexual trophozoite stage is susceptible to iron-induced oxidative stress and is subsequently a target for many of the current anti-malarial drugs. Here, we provide evidence that SUMOylation plays a role in the parasite response to oxidative stress during red blood cell stages, indicative of a protective role seen in other organisms. Using x-ray crystallography, we solved the structure of the human SUMO E1 ubiquitin fold domain in complex with the E2, Ubc9. The interface defined in this structure guided in silico modeling, mutagenesis, and in vitro biochemical studies of the P. falciparum SUMO E1 and E2 enzymes, resulting in the identification of surface residues that explain species-specific interactions. Our findings suggest that parasite-specific inhibitors of SUMOylation could be developed and used in combination therapies with drugs that induce oxidative stress.

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.

Background: Post-translational SUMO modification of TDG weakens its DNA binding and was proposed to regulate dissociation of a tight enzyme-product complex. Results: In vitro sumoylation of TDG by SUMO-1 and SUMO-2 is efficient for free and DNA-bound TDG. Conclusion: E2-mediated sumoylation is not selective for product-bound TDG but could potentially stimulate product release. Significance: Our findings inform the mechanism and role of TDG sumoylation.

No abstract

No abstract