SUMO chain-induced dimerization activates RNF4

Dimeric RING E3 ligases interact with protein substrates and conformationally restrain the ubiquitin-E2-conjugating enzyme thioester complex such that it is primed for catalysis. RNF4 is an E3 ligase containing an N-terminal domain that binds its polySUMO substrates and a C-terminal RING domain responsible for dimerization. To investigate how RNF4 activity is controlled, we increased polySUMO substrate concentration by ablating expression of SUMO protease SENP6. Accumulation of SUMO chains in vivo leads to ubiquitin-mediated proteolysis of RNF4. In vitro we demonstrate that at concentrations equivalent to those found in vivo RNF4 is predominantly monomeric and inactive as an ubiquitin E3 ligase. However, in the presence of SUMO chains, RNF4 is activated by dimerization, leading to both substrate ubiquitylation and autoubiquitylation, responsible for degradation of RNF4. Thus the ubiquitin E3 ligase activity of RNF4 is directly linked to the availability of its polySUMO substrates

RavN is a member of a previously unrecognized group of Legionella pneumophila E3 ubiquitin ligases

The eukaryotic ubiquitylation machinery catalyzes the covalent attachment of the small protein modifier ubiquitin to cellular target proteins in order to alter their fate. Microbial pathogens exploit this post-translational modification process by encoding molecular mimics of E3 ubiquitin ligases, eukaryotic enzymes that catalyze the final step in the ubiquitylation cascade. Here, we show that the Legionella pneumophila effector protein RavN belongs to a growing class of bacterial proteins that mimic host cell E3 ligases to exploit the ubiquitylation pathway. The E3 ligase activity of RavN was located within its N-terminal region and was dependent upon interaction with a defined subset of E2 ubiquitin-conjugating enzymes. The crystal structure of the N-terminal region of RavN revealed a U-box-like motif that was only remotely similar to other U-box domains, indicating that RavN is an E3 ligase relic that has undergone significant evolutionary alteration. Substitution of residues within the predicted E2 binding interface rendered RavN inactive, indicating that, despite significant structural changes, the mode of E2 recognition has remained conserved. Using hidden Markov model-based secondary structure analyses, we identified and experimentally validated four additional L. pneumophila effectors that were not previously recognized to possess E3 ligase activity, including Lpg2452/SdcB, a new paralog of SidC. Our study provides strong evidence that L. pneumophila is dedicating a considerable fraction of its effector arsenal to the manipulation of the host ubiquitylation pathway.Funding: This work was funded by the Intramural Research Program of the National Institutes of Health (to MPM)(Project Number: 1ZIAHD008893-07) and by the Spanish Ministry of Economy and Competitiveness Grant (to AH)(BFU2014-59759-R) and the Severo Ochoa Excellence Accreditation (to AH)(SEV-2016-0644). This study made use of the Diamond Light Source beamline I04 (Oxfordshire, UK) and ALBA synchrotron beamline BL13-XALOC, funded in part by the Horizon 2020 programme of the European Union, iNEXT (H2020 Grant # 653706). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Protein–Protein Interactions Modulate the Docking-Dependent E3-Ubiquitin Ligase Activity of Carboxy-Terminus of Hsc70-Interacting Protein (CHIP)

CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control.Published versio

The activity of TRAF RING homo- and heterodimers is regulated by zinc finger 1

Ubiquitin chains linked through lysine63 (K63) play a critical role in inflammatory signalling. Following ligand engagement of immune receptors, the RING E3 ligase TRAF6 builds K63-linked chains together with the heterodimeric E2 enzyme Ubc13-Uev1A. Dimerisation of the TRAF6 RING domain is essential for the assembly of K63-linked ubiquitin chains. Here, we show that TRAF6 RING dimers form a catalytic complex where one RING interacts with a Ubc13~Ubiquitin conjugate, while the zinc finger 1 (ZF1) domain and linker-helix of the opposing monomer contact ubiquitin. The RING dimer interface is conserved across TRAFs and we also show that TRAF5–TRAF6 heterodimers form. Importantly, TRAF5 can provide ZF1, enabling ubiquitin transfer from a TRAF6-bound Ubc13 conjugate. Our study explains the dependence of activity on TRAF RING dimers, and suggests that both homo- and heterodimers mediated by TRAF RING domains have the capacity to synthesise ubiquitin chains

Regulation of the E3 Ligase Activity of Human Cellular Inhibitor of Apoptosis Proteins

Post-translational modifications serve to regulate the function, activity, abundance or cellular localisation of proteins in a dynamic and timely manner. One form of such modifications involves the covalent attachment of a conserved 76-residue protein called ubiquitin (Ub) to the ε-amino group of Lys side chains. In many cases, Ub attachment (or ‘ubiquitylation’) tags the modified proteins for degradation by the 26S proteasome. Ubiquitylation plays a key role in cell signalling because, by controlling the abundance of specific proteins, it can influence the outcome of certain pathways. Ub attachment requires the activity of three groups of enzymes: E1 Ub-activating enzymes, E2 Ub-conjugating enzymes and E3 Ub-protein ligases. They are often simply referred to as E1 enzymes, E2 enzymes and E3 ligases, respectively. The human genome possesses two genes encoding for E1 enzymes, around 40 genes encoding for E2 enzymes, and more than 600 genes encoding for E3 ligases. The great majority of these E3 ligases possess Really Interesting New Gene (RING) domains, which directly recruit a Ub-loaded E2 enzyme (E2~Ub conjugate) to promote Ub transfer. Several members of the Inhibitor of Apoptosis (IAP) protein family, such as the human cellular IAP 1 and 2 (cIAP1 and cIAP2, respectively), are RING-type E3 ligases. Their E3 ligase activity is essential for regulating pathways that mediate apoptosis, cell division and the immune response. cIAP1 and cIAP2 mainly ubiquitylates components of the Tumour Necrosis Factor α (TNFα) Receptor 1 (TNF-R1)-associated protein complex. This process leads to stabilisation of the complex and promotes the expression of anti-apoptotic genes. The realisation that many cancer cell lines over-express cIAP1 and cIAP2 to promote their survival has sparked the development of a new class of drugs named SMAC-mimetic (SM) compounds. Binding of SM compounds to the Baculoviral IAP Repeat 3 (BIR3) domain of cIAP1 and cIAP2 has been shown to promote their self-ubiquitylation (or ‘auto-ubiquitylation’) and proteasomal degradation, as well as sensitise the cells to other killing agents. Studies by previous members of our group have shown that dimerisation of the C-terminal RING domain of cIAP2 is essential for ubiquitylation. Using purified recombinant cIAP1 and cIAP2 proteins, this study revealed that cIAP1 adopted a predominantly monomeric conformation in solution. This was in contrast to the more dimeric cIAP2. Measurements using a number of biophysical methods also showed that the addition of SM compounds promoted RING domain-dependent dimerisation of cIAP1. Dimerisation was not accompanied by changes in the secondary structural contents, suggesting that SM compounds only promoted domain re-arrangements. Furthermore, subsequent activity assays pointed to a role of the BIR3 domain in inhibiting RING domain dimerisation. This first part of the project highlights the importance of a non-catalytic (BIR3) domain in regulating the activity and oligomeric state of the catalytic RING domain. In addition, the results provide insights into the mechanism of SM compound-induced activation of cIAP1. In addition to the BIR3 and RING domains, cIAP1 and cIAP2 also possesses a Ub-binding domain called the Ubiquitin-Associated (UBA) domain. Others have suggested that the UBA domain had important roles in the induction of anti-apoptotic genes, and/or the proteasomal degradation of cIAP1 and cIAP2 following auto-ubiquitylation. However, these studies did not find any evidence for the role of the UBA domain in influencing E3 ligase activity. It should be noted that, in these studies, Ala substitution was introduced to the conserved motif of the UBA domain. Herein, it is demonstrated that this commonly-introduced mutation destabilises the UBA domain fold and causes artificial alterations in the E3 ligase activity pattern of the resulting mutant cIAP1 protein. With this in mind, a new fully-folded UBA domain mutant cIAP1 protein displaying impaired Ub binding was generated. E3 ligase activity comparison between wild-type cIAP1 and the new mutant protein revealed that the UBA domain functioned to accelerate the initial rate of auto- and substrate ubiquitylation. This rate enhancement was likely caused by direct E2~Ub conjugate recruitment by the UBA domain. Interestingly, similar observations were also made using cIAP2, suggesting that the mechanism is conserved. Overall, this part of the project uncovered an additional layer of E3 ligase activity regulation in cIAP1 and cIAP2, which has been previously overlooked