Regulation of ubiquitin transfer by XIAP, a dimeric RING E3 ligase

2013 ◽  
Vol 450 (3) ◽  
pp. 629-638 ◽  
Author(s):  
Yoshio Nakatani ◽  
Torsten Kleffmann ◽  
Katrin Linke ◽  
Stephen M. Condon ◽  
Mark G. Hinds ◽  
...  
Keyword(s):  
Biochemical Analysis ◽  
E3 Ligase ◽  
Ring Domain ◽  
Aromatic Residue ◽  
E3 Ligases ◽  
Dimer Interface ◽  
Ring E3 Ligase ◽  
Thioester Bond ◽  
Ring E3 ◽  
Apoptosis Proteins

RING domains of E3 ligases promote transfer of Ub (ubiquitin) from the E2~Ub conjugate to target proteins. In many cases interaction of the E2~Ub conjugate with the RING domain requires its prior dimerization. Using cross-linking experiments we show that E2 conjugated ubiquitin contacts the RING homodimer interface of the IAP (inhibitor of apoptosis) proteins, XIAP (X-linked IAP) and cIAP (cellular IAP) 2. Structural and biochemical analysis of the XIAP RING dimer shows that an aromatic residue at the dimer interface is required for E2~Ub binding and Ub transfer. Mutation of the aromatic residue abolishes Ub transfer, but not interaction with Ub. This indicates that nuleophilic attack on the thioester bond depends on precise contacts between Ub and the RING domain. RING dimerization is a critical activating step for the cIAP proteins; however, our analysis shows that the RING domain of XIAP forms a stable dimer and its E3 ligase activity does not require an activation step.

scholarly journals A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation

Science ◽  
2019 ◽  
Vol 365 (6448) ◽  
pp. eaaw4912 ◽  
Author(s):  
Richard T. Timms ◽  
Zhiqian Zhang ◽  
David Y. Rhee ◽  
J. Wade Harper ◽  
Itay Koren ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Stability ◽  
Protein Degradation ◽  
E3 Ligase ◽  
Cleavage Sites ◽  
Caspase Cleavage ◽  
E3 Ligases ◽  
Ring E3 Ligase ◽  
The Stability ◽  
Ring E3

The N-terminal residue influences protein stability through N-degron pathways. We used stability profiling of the human N-terminome to uncover multiple additional features of N-degron pathways. In addition to uncovering extended specificities of UBR E3 ligases, we characterized two related Cullin-RING E3 ligase complexes, Cul2ZYG11B and Cul2ZER1, that act redundantly to target N-terminal glycine. N-terminal glycine degrons are depleted at native N-termini but strongly enriched at caspase cleavage sites, suggesting roles for the substrate adaptors ZYG11B and ZER1 in protein degradation during apoptosis. Furthermore, ZYG11B and ZER1 were found to participate in the quality control of N-myristoylated proteins, in which N-terminal glycine degrons are conditionally exposed after a failure of N-myristoylation. Thus, an additional N-degron pathway specific for glycine regulates the stability of metazoan proteomes.

scholarly journals Intracellular antibody signalling is regulated by phosphorylation of the Fc receptor TRIM21

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Claire Dickson ◽  
Adam J Fletcher ◽  
Marina Vaysburd ◽  
Ji-Chun Yang ◽  
Donna L Mallery ◽  
...  
Keyword(s):  
Cell Surface ◽  
Binding Site ◽  
Immune Activation ◽  
Rna Viruses ◽  
E3 Ligase ◽  
Ring Domain ◽  
Ring E3 Ligase ◽  
Intracellular Antibody ◽  
Ring E3 ◽  
Cytokine Transcription

Cell surface Fc receptors activate inflammation and are tightly controlled to prevent autoimmunity. Antibodies also simulate potent immune signalling from inside the cell via the cytosolic antibody receptor TRIM21, but how this is regulated is unknown. Here we show that TRIM21 signalling is constitutively repressed by its B-Box domain and activated by phosphorylation. The B-Box occupies an E2 binding site on the catalytic RING domain by mimicking E2-E3 interactions, inhibiting TRIM21 ubiquitination and preventing immune activation. TRIM21 is derepressed by IKKβ and TBK1 phosphorylation of an LxxIS motif in the RING domain, at the interface with the B-Box. Incorporation of phosphoserine or a phosphomimetic within this motif relieves B-Box inhibition, promoting E2 binding, RING catalysis, NF-κB activation and cytokine transcription upon infection with DNA or RNA viruses. These data explain how intracellular antibody signalling is regulated and reveal that the B-Box is a critical regulator of RING E3 ligase activity.

scholarly journals Ubiquitin transfer by a RING E3 ligase occurs from a closed E2~Ub conformation

2019 ◽  
Author(s):  
Emma Branigan ◽  
J. Carlos Penedo ◽  
Ronald T. Hay
Keyword(s):  
Single Molecule ◽  
Active Form ◽  
Resonance Energy Transfer ◽  
E3 Ligase ◽  
Resonance Energy ◽  
Post Translational Modification ◽  
E3 Ligases ◽  
Closed Conformation ◽  
Ring E3 Ligase ◽  
Ring E3

AbstractUbiquitination is a eukaryotic post-translational modification that modulates a host of cellular processes1. Modification is mediated by an E1 activating enzyme (E1), an E2 conjugating enzyme (E2) and an E3 ligase (E3). The E1 catalyses formation of a highly reactive thioester linked conjugate between ubiquitin and E2 (E2~Ub)2. The largest class of ubiquitin E3 ligases, which is represented by RING E3s, bind both substrate and E2~Ub and facilitate transfer of ubiquitin from the E2 to substrate. Based on extensive structural analysis3–5 it has been proposed that RING E3s prime the E2~Ub conjugate 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 a substrate nucleophile. However the proposal that the RING dependent closed conformation of E2~Ub represents the active form that mediates ubiquitin transfer is a model that has yet to be experimentally tested. Here we use single molecule Förster Resonance Energy Transfer (smFRET) to test this hypothesis and demonstrate that ubiquitin is transferred from the closed conformation during an E3 catalysed reaction. Using Ubc13 as an E2, we designed a FRET labelled E2~Ub conjugate, which distinguishes between closed and alternative conformations. Firstly, we defined the high FRET state as the closed conformation using a stable isopeptide linked E2~Ub conjugate, while the low FRET state represents more open conformations. Secondly, we developed a real-time smFRET assay to monitor RING E3 catalysed ubiquitination with a thioester linked E2~Ub conjugate and determined the catalytically active conformation. Our results demonstrate that the reaction proceeds from the high FRET or closed conformation and confirm the hypothesis that the closed conformation is the active form of the conjugate. These findings are not only relevant to RING E3 catalysed ubiquitination but are also broadly applicable to E3 mediated ligation of other ubiquitin-like proteins (Ubls) to substrates.

scholarly journals RING domain dimerization is essential for RNF4 function

2010 ◽  
Vol 431 (1) ◽  
pp. 23-29 ◽  
Author(s):  
Chu Wai Liew ◽  
Huaiyu Sun ◽  
Tony Hunter ◽  
Catherine L. Day
Keyword(s):  
Ubiquitin Ligases ◽  
Ring Domain ◽  
E3 Ligases ◽  
Finger Protein ◽  
Thioester Bond ◽  
New Gene ◽  
The Stability ◽  
Ring E3 Ligases ◽  
Ring E3

RNF4 [RING (really interesting new gene) finger protein 4] family ubiquitin ligases are RING E3 ligases that regulate the homoeostasis of SUMOylated proteins by promoting their ubiquitylation. In the present paper we report that the RING domain of RNF4 forms a stable dimer, and that dimerization is required for ubiquitin transfer. Our results suggest that the stability of the E2~ubiquitin thioester bond is regulated by RING domain dimerization.

scholarly journals Structural insights into the nanomolar affinity of RING E3 ligase ZNRF1 for Ube2N and its functional implications

2018 ◽  
Vol 475 (9) ◽  
pp. 1569-1582 ◽  
Author(s):  
Adaitya Prasad Behera ◽  
Pritam Naskar ◽  
Shubhangi Agarwal ◽  
Prerana Agarwal Banka ◽  
Asim Poddar ◽  
...  
Keyword(s):  
Dissociation Constants ◽  
E3 Ligase ◽  
E3 Ligases ◽  
Ring E3 Ligase ◽  
New Gene ◽  
Functional Analyses ◽  
Affinity Interaction ◽  
Ring E3 Ligases ◽  
Ring E3 ◽  
Structural Insights

RING (Really Interesting New Gene) domains in ubiquitin RING E3 ligases exclusively engage ubiquitin (Ub)-loaded E2s to facilitate ubiquitination of their substrates. Despite such specificity, all RINGs characterized till date bind unloaded E2s with dissociation constants (Kds) in the micromolar to the sub-millimolar range. Here, we show that the RING domain of E3 ligase ZNRF1, an essential E3 ligase implicated in diverse cellular pathways, binds Ube2N with a Kd of ∼50 nM. This high-affinity interaction is exclusive for Ube2N as ZNRF1 interacts with Ube2D2 with a Kd of ∼1 µM, alike few other E3s. The crystal structure of ZNRF1 C-terminal domain in complex with Ube2N coupled with mutational analyses reveals the molecular basis of this unusual affinity. We further demonstrate that the ubiquitination efficiency of ZNRF1 : E2 pairs correlates with their affinity. Intriguingly, as a consequence of its high E2 affinity, an excess of ZNRF1 inhibits Ube2N-mediated ubiquitination at concentrations ≥500 nM instead of showing enhanced ubiquitination. This suggests a novel mode of activity regulation of E3 ligases and emphasizes the importance of E3-E2 balance for the optimum activity. Based on our results, we propose that overexpression-based functional analyses on E3 ligases such as ZNRF1 must be approached with caution as enhanced cellular levels might result in aberrant modification activity.

scholarly journals Structure and dynamics of the ASB9 CUL-RING E3 Ligase

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ryan J. Lumpkin ◽  
Richard W. Baker ◽  
Andres E. Leschziner ◽  
Elizabeth A. Komives
Keyword(s):  
E3 Ligase ◽  
Structure And Dynamics ◽  
Ring E3 Ligase ◽  
Ring E3
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